miR-192, miR-194 and miR-215: a convergent microRNA network suppressing tumor progression in renal cell carcinoma

For Peer Review

miR-192, miR-194 and miR-215: A Convergent miRNA

Network Suppressing Tumor Progression in Renal Cell

Carcinoma

Journal: Carcinogenesis

Manuscript ID: CARCIN-2013-00051.R2

Manuscript Type: Original Manuscript

Date Submitted by the Author: 22-May-2013

Complete List of Authors: Khella, Heba; Li Ka Shing Knowledge Institute of St. Michael’s Hospital,

Department of Laboratory Medicine and the Keenan Research Centre Bakhet, Marize; Li Ka Shing Knowledge Institute of St. Michael’s Hospital, Department of Laboratory Medicine and the Keenan Research Centre Allo, Ghassan; University of Toronto, Department of Laboratory Medicine and Pathobiology Jewett, Michael; 4. Division of Urologic Oncology, Princess Margaret Hospital, University Health Network, University of Toronto, Department of Surgery Girgis, Andrew; Li Ka Shing Knowledge Institute of St. Michael’s Hospital, Department of Laboratory Medicine and the Keenan Research Centre Latif, Ashraf; Li Ka Shing Knowledge Institute of St. Michael’s Hospital, Department of Laboratory Medicine and the Keenan Research Centre

Girgis, Hala; Li Ka Shing Knowledge Institute of St. Michael’s Hospital, Department of Laboratory Medicine and the Keenan Research Centre Von Both, Ingo; University of Toronto, Department of Laboratory Medicine and Pathobiology Bjarnason, Georg; Sunnybrook Odette Cancer Center, 5. Division of Medical Oncology and Hematology Yousef, GM; Li Ka Shing Knowledge Institute of St. Michael’s Hospital, Department of Laboratory Medicine and the Keenan Research Centre ; University of Toronto, Institute of Medical Science; University of Toronto, Department of Laboratory Medicine and Pathobiology

Keywords: Renal cell carcinoma, miRNA, Tumor Markers, Personalized Medicine, Targeted Therapy

Carcinogenesis Carcinogenesis Advance Access published May 28, 2013 by guest on O

ctober 15, 2016http://carcin.oxfordjournals.org/

Dow

nloaded from

http://carcin.oxfordjournals.org/

For Peer Review

1

miR-192, miR-194 and miR-215: A Convergent miRNA Network Suppressing

Tumor Progression in Renal Cell Carcinoma

Heba WZ Khella1, 2

, Marize Bakhet1, Ghassan Allo

3, Michael AS Jewett

4, Andrew H

Girgis1, Ashraf Latif

1, Hala Girgis

1, Ingo Von Both

3, Georg A Bjarnason

5, George M

Yousef1,2, 3,ж

1. Department of Laboratory Medicine and the Keenan Research Centre in the Li Ka

Shing Knowledge Institute of St. Michael’s Hospital, Toronto, ON, M5B 1W8,

Canada.

2. Institute of Medical Science, University of Toronto, Toronto, ON, M5S 1A8, Canada

3. Department of Laboratory Medicine and Pathobiology, University of Toronto,

Toronto, ON, M5S 1A8, Canada.

4. Division of Urologic Oncology, Princess Margaret Hospital, University Health

Network, Department of Surgery, University of Toronto, Toronto, ON, M5G 2M9,

Canada

5. Division of Medical Oncology and Hematology, Sunnybrook Odette Cancer Center,

Toronto, ON, M4N 3M5, Canada

ж To whom correspondence should be addressed

Running Title: microRNA in renal cell carcinoma

Corresponding Author:

George M Yousef, MD PhD FRCPC (Path)

Department of Laboratory Medicine,

St. Michael's

30 Bond Street, Toronto, ON, M5B 1W8, Canada

Tel: +1-416-864-6060 Ext: 77605

Fax: 416-864-5648

E-Mail: [email protected]

Page 1 of 66 Carcinogenesis

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

by guest on October 15, 2016

http://carcin.oxfordjournals.org/D

ownloaded from


For Peer Review

2

Abstract

miRNAs play a crucial role in tumor progression and metastasis. We and others

recently identified a number of miRNAs that are dysregulated in metastatic, compared to

primary renal cell carcinoma (RCC). Here, we investigated three miRNAs that are

significantly downregulated in metastatic tumors; miR-192, miR-194 and miR-215. Gain-

of-function analyses showed that restoration of their expression decreases cell migration

and invasion in RCC cell line models, while knockdown of these miRNAs resulted in

enhancing cellular migration and invasion abilities. We identified three targets of these

miRNAs with potential role in tumor aggressiveness; MDM2, TYMS, and SIP1/ZEB2.

We observed a convergent (the same molecule can be targeted by all three miRNAs) and

a divergent (the same miRNA can control multiple targets) effects for these miRNAs. We

experimentally validated these miRNA-target interactions using three independent

approaches. First, we observed that miRNA overexpression significantly reduces the

mRNA and protein levels of their targets. In the second, we observed significant

reduction of the luciferase signal of a vector containing the 3’UTR of the target upon

miRNA overexpression. Finally, we show the presence of inverse correlation between

miRNA changes and the expression levels of their targets in patient specimens. We also

examined the prognostic significance of miR-215 in RCC. Lower expression of miR-215

is associated with significantly reduced disease-free survival time. These findings were

validated on an independent dataset from The Cancer Genome Atlas. These results can

pave the way to the clinical use of miRNAs as prognostic markers and therapeutic

targets.

Page 2 of 66Carcinogenesis

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960



ownloaded from


For Peer Review

3

Keywords

Renal cell carcinoma; miRNA; tumor markers; metastasis; prognosis; miR-192; miR-

194; miR-215; kidney cancer; personalized medicine; targeted therapy.

Non-standard abbreviations

ccRCC, clear cell renal cell carcinoma; EMT, epithelial to mesenchymal; miRNA,

microRNA ; qRT-PCR, quantitative reverse transcription polymerase chain reaction;

SIP1/ZEB2, Smad Interacting protein 1/zinc finger E-box binding homeobox 2; MDM2,

murine double minute 2; TYMS, thymidylate synthase ; HIF-1α, hypoxia inducible factor

1 alpha subunit; PPIA, Peptidylprolyl isomerase A (cyclophilin A); HPRT1,

hypoxanthine phosphoribosyltransferase 1; 5-FU, 5-fluorouracil


123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960



ownloaded from


For Peer Review

4

Introduction

Renal cell carcinoma (RCC) accounts for about 90% of the adult kidney cancers,

[1] and is one of the top ten prevalent cancers in North America. The incidence of RCC is

steadily rising in the past few decades [2]. It is also an aggressive tumor with 35% overall

mortality and 30% metastatic potential. Favorable prognosis of RCC is associated with

early diagnosis and treatment while patients diagnosed at the metastatic stage have poor

prognosis with only 9% 5-year survival rate. 90% of cancer associated mortality in RCC

is due to metastasis [3]. Unfortunately, there are no biomarkers available to accurately

predict the prognosis of RCC. Therefore, there is an urgent need for more understanding

of the pathogenesis of RCC metastasis as a crucial step towards identification of

prognostic markers and development of targeted therapies for this aggressive tumor.

miRNAs are short non-coding RNAs that regulate gene expression by binding to

their target genes. They regulate critical biological processes including development, cell

differentiation, proliferation and apoptosis [4]. They also play important roles in tumor

development and metastasis [5]. miRNAs were found to be differentially expressed in

cancer, and were shown to target key molecules involved in tumor progression [6].

miRNA dysregulation in RCC is recently reported [7;8], and their involvement in RCC

pathogenesis is also documented [9-12]. They have potential utilities as cancer

biomarkers and therapeutic targets [13;14].

We, and others, identified a number of miRNAs that are differentially expressed

in metastatic RCC compared to primary tumors [15-17]. miR-192, miR-194 and miR-215


123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960



ownloaded from


For Peer Review

5

were among the most significantly downregulated in metastatic ccRCC. These three

miRNAs are highly enriched in the normal kidney [18], and miR-192 and miR-194 were

reported to be strongly expressed in renal cortex [19]. All three miRNAs can be induced

by p53 [20] and they are also reported to be p53 positive regulators through an

autoregulatory loop [21]. A recent study reported the role of these miRNAs in

endometrial cancer progression, and suggested their potential therapeutic utility [22].

Also, miR-194 overexpression is reported to suppress liver cancer metastasis.

The three miRNAs are found in two clusters, the miR-215/miR-194-1 cluster on

chromosome 1 (1q41) and the miR-192/miR-194-2 cluster on chromosome 11 (11q13.1).

miR-194-1 and miR-194-2 have the same mature sequence that are derived from two

different precursors on two chromosomal locations. miR-192 and miR-215 are closely

related with similar seed sequence.

In this study, we delineate the functional involvement of miR-192, miR-194 and

miR-215 in RCC progression. We provide experimental evidence that these miRNAs

affect cell migration and invasion abilities. We also identified three targets of these

miRNAs that are related to cancer aggressiveness; MDM2, TYMS, and SIP1/ZEB2. We

experimentally validated the miRNA-target interactions using three independent

approaches in vitro and in vivo. We finally provide preliminary evidence on the potential

significance of miR-215 as a prognostic marker in RCC. Our findings document the

active involvement of miRNAs in kidney cancer progression. We also uncover a miRNA

network with convergent properties (where multiple miRNAs target the same molecule).


123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960



ownloaded from


For Peer Review

6

We further document the presence of a divergent effect of these miRNAs with the same

miRNA being able to simultaneously control a number of targets.


123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960



ownloaded from


For Peer Review

7

Materials and methods

Specimen collection

20 Primary ccRCC cancerous tissue specimens and 61 formalin-fixed paraffin-

embedded tissues were collected from St. Michael’s Hospital, and the Ontario Tumor

Bank, Toronto, Canada. Expression data from additional 218 patients with primary

ccRCC were obtained from The Cancer Genome Atlas (TCGA) Database. Fresh

specimens were collected immediately after resection, snap frozen in liquid nitrogen, and

stored at -80ºC until total RNA extraction. Areas of pure tumor tissues were identified by

a pathologist. Samples were taken from areas with no hemorrhage or necrosis and

multiple sections were mixed from the same tumor to compensate for tumor

heterogeneity. All the procedures were approved by the Research Ethics Board at St.

Michael’s Hospital, Toronto, Canada

RNA Extraction and quantitative RT-PCR (qRT-PCR)

Two mg of fresh frozen ccRCC tissues were used for nucleic acid isolation. For

formalin-fixed tumors, nucleic acid isolation was done using 6 cores of pure tumor areas

from formalin-fixed paraffin-embedded tissues of ccRCC (each core was 1.0 mm in

diameter). Total RNA was extracted using the miRNeasy Kit (Qiagen, Mississauga,

Canada) according to the manufacture’s protocol. RNA quality and concentration were

determined spectrophotometrically.

For miRNA analyses, miRNA-specific reverse transcription was performed with

5ng total RNA using the TaqMan® MicroRNA Reverse Transcription Kit (Applied


123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960



ownloaded from


For Peer Review

8

Biosystems, Foster City, CA) as described by the manufacture for miR-192, miR-194,

and miR-215. Thermal cycling conditions are shown in Supplementary Table (1-A).

qRT-PCR was performed using the TaqMan microRNA Assay® Kit on the Step

One™ Plus Real-Time PCR System (Applied Biosystems, Foster City, CA). Thermal

cycling conditions were according to the manufacture’s fast protocol and all reactions

were performed in triplicate. Relative expression was determined using the ∆∆CT method

and expression values were normalized to small nuclear RNA, RNU48 (Applied

Biosystems, Foster City, CA) which was shown to be stably expressed in ccRCC tissues

[23]. Thermal cycling conditions are shown in Supplementary Table (1-B).

For the expression analyses of target genes, the primer sequence are shown in

Supplementary Table (2)

Reverse transcription was preformed with High capacity RNA-to-cDNA kit

(Applied Biosystems, Foster City, CA) as per the manufacture’s instructions. Thermal

cycling conditions are shown in Supplementary Table (1-C).

qRT-PCR was performed using the Fast Syber Green Master Mix (Applied

Biosystems, Foster City, CA). Peptidylprolyl isomerase A (cyclophilin A) (PPIA) or

hypoxanthine phosphoribosyltransferase 1 (HPRT1) were used as endogenous controls.

Thermal cycling conditions are shown in Supplementary Table (1-D).


123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960



ownloaded from


For Peer Review

9

Cell culture and miRNA transfection

786-O, ACHN and CAKI-1 renal cell carcinoma cell lines were obtained from

American Type Culture Collection (ATCC; Manassas, VA) and were grown according to

manufacturer’s protocol. Pre-miR™ precursors and anti-miR™ miR inhibitors for miR-

192, miR-194 and miR-215 were purchased from Applied Biosystems (Foster City,

USA). Cells were transfected using siPORT™ NeoFX™ transfection agent (Ambion,

Austin, TX) as recommended by the manufacture and described in our previous

publications [24;25]. Briefly, transfection agent was diluted in Opti-MEM® Reduced

Serum Media (Invitrogen, Carlsbad, USA) and incubated for 10 minutes at room

temperature. miRNA precursors and inhibitors were diluted in the same Media to a final

concentration of 30nM, and then combined with transfection agent and incubated for 10

minutes at room temperature. Transfection mixtures were added to the cell culture plate

and overlaid with cell suspensions. Cells were then incubated at 37ºC and 5% CO2. Three

separate transfections were performed and each was analyzed in triplicate. Transfection

efficiency was confirmed using BLOCK-IT Fluorescent Oligo (Invitrogen)

Migration assay

786-O cells were seeded in a 12-well plate, and transfected either with SiPORT™

NeoFX™ transfection agent, scrambled miRNA, miR-192, miR-194 and miR-215 or

their inhibitors, or co-transfected with the miRNA and its inhibitor. Twenty four hours

after transfection, the cell monolayer was wounded using a 200µL pipette tip.

Hydroxyurea 100mM was added to the cell culture to inhibit cell proliferation.

Photomicrographs were taken every 30 minutes starting at the time of wounding (0 hrs)


123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960



ownloaded from


For Peer Review

10

and continued up to 9 hrs using a microscope in an incubation chamber with 37ºC and

5% CO2. This microscope was programmed to take a series of photomicrographs at the

exact place. Image J Software (National Institutes of Health, Bethesda, MD, USA,

http://rsbweb.nih.gov/ij/) was used for cell migration analysis. Percent cell-free area was

calculated as [(cell-free area9hrs/cell-free area0hrs) x 100] and cell migration rate was

displayed as the percent of cell covered area (100 - percent cell-free area). Each

experiment was performed in triplicate.

Invasion Assay

The effect of miR-192, miR-194 and miR-215 on cellular invasion were examined

using BD BioCoat Matrigel Invasion Chamber (BD Biosciences, Bedford, MA). 786-O

and ACHN cells were transfected either with SiPORT™ NeoFX™ transfection agent,

scrambled miRNA, miR-192, miR-194, miR-215 or their inhibitors, or co-transfected

with the miRNA and its inhibitor. Twenty four hours later, cells were trypsinized and

resuspended in low serum media (DMEM supplemented with 0.5% FBS). DMEM

supplemented with 10% FBS was used as a chemoattractant, added to the bottom

chamber, and cells (5.0 x 104

cells/ml) were plated on the upper chamber. Cells were

incubated for 22 hours at 37ºC and 5% CO2 in a humidified tissue culture incubator.

After incubation, non-invading cells were removed from the upper surface of the

membrane and cells on the lower surface were stained with Diff-Quick. The experiment

was performed in triplicate. Photomicrographs at 40X magnification in three fields were

taken and invading cells were counted. The percentage invasion was calculated [(Mean


123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960



ownloaded from


For Peer Review

11

number of cells that invaded through Matrigel insert/ Mean number of cells that migrated

through the control insert membrane) x 100].

Cell proliferation assay

Cellular proliferation was measured by the 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-

diphenyltetrazolium bromide (MTT; Roche, Mississauga, ON) assay. Cells were plated at

6.0 x 103 cells per well in a 96 well plate and transfected either with SiPORT™ NeoFX™

transfection agent, miR-192, miR-194 and miR-215 or co-transfected with the miRNA

and its inhibitor. Cells were incubated for 3 days after which 10µL of 5 mg/mL solution

of MTT in phosphate buffered saline (PBS) was added and incubated for 4 hours at 37°C.

The precipitate was then solubilized in 100µL solubilization solution (10% SDS in 0.01

M HCl) and incubated at 37°C overnight. The absorbance of each well was measured at a

wavelength of 550nm. Each test was repeated in triplicate.

Western Blot

CAKI-1 cells were transfected either with miR-192, miR-194, or miR-215 or co-

transfected with the miRNA and its inhibitor. 48 hours later, cells were lysed using

NETN lysis buffer (pH 8.0) with protease inhibitor cocktail tablets (Roche, Mississauga,

Canada). Proteins were collected after centrifugation at 21,000 x g for 10 minutes at 4°C.

BCA Protein Assay Reagent (Pierce Biotechnology, Rockford, IL) was used to determine

protein concentrations. Total cellular proteins were separated in a 10% sodium dodecyl

sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a

nitrocellulose membrane. The membrane was blocked with 5% BSA or 5% milk in TBST


123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960



ownloaded from


For Peer Review

12

and incubated with primary antibodies diluted in blocking solution overnight at 4ºC with

shaking. The membranes were then washed with TBST, incubated with appropriate

secondary antibody diluted in blocking solution for one hour. The following antibodies

were used in this study anti-Thymidylate Synthase (Millipore Corporation), MDM2

antibody (Santa Cruz Biotechnology Inc.) and anti-SIP1/ ZEB2 (Abcam, Camridge, MA,

USA). Membranes were stripped and re-probed for anti-α-tubulin (Cell Signalling

Technology Inc.) as a loading control. Western blot photos were taken and quantified

using the VersaDoc Imaging System (Bio-Rad, Hercules, CA), QuantityOne and Image

Lab 3.0 programs.

Luciferase assay

Luciferase reporter plasmids containing the 3’ UTR of TYMS, MDM2 and ZEB2

were obtained from SwitchGear Genomics (Menlo Park, CA). Empty vector was used as

a positive control. 786-O cells were seeded in 96-well plates. On the second day, cells

were transfected with the Luciferase reporter plasmids containing the 3’UTR of TYMS,

MDM2 and ZEB2, the empty vector or co-transfected with the plasmids and miRNAs

(miR-192, miR-194 or miR-215) using the DharmaFect Duo transfection agent

(Dharmacon, Thermoscientific) as recommended by the manufacture. On the third day,

luciferase activity was measured using the LightSwitch Luciferase Assay Reagents

(SwitchGear Genomics, Menlo Park, CA). Luminescence was calculated for each

construct (luminescence = specific miRNA/non-targeting control).


123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960



ownloaded from


For Peer Review

13

Survival Analysis

miR-215 expression was measured using qRT-PCR in 61 formalin-fixed paraffin-

embedded (FFPE) tissues of primary ccRCC. Survival analysis was performed by

constructing Kaplan-Meier disease-free survival (DFS) curve. Disease-free survival was

defined as the time between the initial resection of the kidney tumor and the occurrence

of recurrence or metastasis.

Bioinformatic analysis

‘Level 3’ miRNA expression data (normalized gene expression generated using

the Illumina GA miRNASeq platform) for miR-215 in 218 patients with primary ccRCC

and overall survival data were obtained from The Cancer Genome Atlas (TCGA). The X-

tile algorithm [26] was used to generate a prognostic optimal cutoff point to dichotomize

miR-215 miRNA expression as ‘miR-215 – High Expression’ and ‘miR-215 – Low

Expression’ using the lowest Monte Carlo p value <0.05. TCGA data types, platforms,

and methodologies are as described previously (The Cancer Genome Atlas Research

Network 2008).

Phylogenetic analysis

The University of California Santa Cruise (UCSC) Genome Browser was used for

sequence comparison of miR-192, miR-194 and miR-215. Conservation among species

of these miRNAs was examined with sequence alignment in the genomes of 28 vertebrate

species, including 17 mammalian species.


123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960



ownloaded from


For Peer Review

14

Results

miR-192, miR-194 and miR-215 overexpression decrease cellular migration rate

We first checked the endogenous expression levels of miR-192, miR-194 and

miR-215 in a number of kidney cancer cell lines. The 786-O, ACHN and CAKI-1 cell

lines were found to have very low expression (compared to a pool of patient primary

ccRCC tumor tissues) (Supplementary Figure 1). The cells were then transfected with

each of these miRNAs. Successful transfection was confirmed by qRT-PCR by

comparing the pre to post transfection levels of the miRNA (Supplementary Figure 2).

The efficiency of the anti-miR decreased when the cell doubled. However, the cells

maintained enough levels during the experimental procedures (up to 3 days).

Next, we investigated the effect of these miRNAs on cell migration using wound

healing assay. The 786-O cells were transfected with miR-192, miR-194, miR-215, or

their inhibitors, or co-transfected with each of these miRNAs and its inhibitor. A number

of controls were also used including untransfected cells, cells transfected with

transfection agent only, a miRNA with no effect on migration (as a negative control), and

cells transfected with positive control (a miRNA with known effect on migration).

Transfection with each of the three miRNAs resulted in significant reduction in the rate

of cell migration compared to controls. As shown in Figure 1A and 1B, overexpression

of miR-192 significantly reduced cellular migration compared to untransfected cells, cells

transfected with transfection reagent only (p = 0.0045), anti-miR-192 (p = 0.0061), or

scrambled miRNA control (p = 0.0094). Also, miR-194 significantly slowed cellular

migration compared to control cells transfected with transfection agent only or with anti-


123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960



ownloaded from


For Peer Review

15

miR-194 (p = 0.0105 and p = 0.0196, respectively) (Figure 1C). The same was observed

for miR-215, where transfected cells showed significantly reduced cell migration rate

compared to transfection reagent control (p = 0.0143) (Figure 1D). The co-transfection

of the miRNA and its inhibitor resulted in restoration of the rate of cellular migration,

further confirming our results (Figure 1). We were not able to identify other cell lines

with overexpression of these miRNAs and thus, knockdown experiments were not

performed.

miR-192, miR-194 and miR-215 overexpression reduce cellular invasion

We also examined the effect of miR-192, miR-194 and miR-215 on cellular invasion

ability using 786-O and ACHN cell line models. Appropriate controls were used as

above. Cells transfected with miR-192, miR-194 or miR-215 showed significant decrease

in cellular invasion compared to untransfected cells, cells transfected with transfection

agent only, scrambled miRNA or each of these miRNAs inhibitor. The rate of cellular

invasion was partially restored when co-transfecting the miRNA and its inhibitor (Figure

2 & Supplementary Figure 3).

miR-192, miR-194 and miR-215 overexpression have insignificant effects on cellular

proliferation

We assessed the effect of miR-192, miR-194 and miR-215 on cellular

proliferation. Three kidney cancer cell lines; ACHN, CAKI-1 and 786-O, were

transfected with miR-192, miR-194, or miR-215. These three miRNAs showed little


123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960



ownloaded from


For Peer Review

16

insignificant reduction of cellular proliferation in ACHN cells (Supplementary Figure

4). There was no significant effect on cell proliferation in CAKI-1 and 786-0 cell lines.

miR-192, miR-194 and miR-215 can target ZEB2, MDM2 and TYMS

To elucidate the mechanisms by which 192, miR-194 and miR-215 can affect

cellular processes involved in metastasis as cellular migration and invasion; we

performed target prediction analysis and identified a number of pathways and targets that

are known to be involved in tumor progression (Supplementary Table 3). It is worth

mentioning that targets of these three miRNAs are overlapping because of the high

degree of similarity in the seed sequence of the miRNAs. We selected three potential

targets; Murine double minute 2 (MDM2), Thymidylate Synthetase (TYMS), and Smad

Interacting protein 1/zinc finger E-box binding homeobox 2 (SIP1/ZEB2) for

experimental validation. These molecules are reported in the literature to contribute to

tumor progression and metastasis.

We experimentally validated the miRNA-target interactions using three

independent approaches. The first is by measuring the effect of miRNA overexpression

on mRNA and protein levels of the predicted target. The second approach was to measure

the effect of miRNA overexpression on a luciferase signal of a vector containing the

3’UTR of the predicted target. The third is to examine the presence of inverse correlation

between miRNA expression and the expression levels of their targets in vivo in patient

tissues.


123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960



ownloaded from


For Peer Review

17

In the first approach, we examined the effect of overexpression of these three

miRNAs on the expression of the targets. We screened a number of kidney cancer cell

lines and identified three RCC cell line models (786-O, ACHN and CAKI-1) with high

endogenous expression levels of ZEB2, MDM2 and TYMS (Supplementary Figure 5).

We then compared the level of expression of these targets; at both the mRNA and protein

levels, before and after transfection of each of these miRNAs.

At the mRNA level, overexpression of each of miR-192, miR-194, and miR-215

significantly decrease ZEB2 expression in 786-O (Figure 3A-C), CAKI

(Supplementary Figure 6A) and ACHN cell lines (Supplementary Figure 6B)

compared to control cells. Co-transfection of these cells with the miRNAs and their

inhibitors was able to restore ZEB2 expression to almost normal levels.

We also validated miRNA-TYMS interactions in CAKI-1, 786-O and ACHN

cells. Overexpression of any of the three miRNAs resulted in significant reduction in

TYMS expression, at the mRNA level, in CAKI-1 (Supplementary Figure 7A-C), 786-

O (Supplementary Figure 7D) and ACHN cells (Supplementary Figure 7E). The

most significant drop in the level of TYMS was seen with miR-192. Transfection with

either miR-194 or miR-215 caused less reduction of TYMS levels, although still

statistically significant.

Similarly, MDM2 expression was significantly decreased after miR-192, miR-

194, or miR-215 overexpression in ACHN (Supplementary Figure 8A-C), CAKI-1 and


123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960



ownloaded from


For Peer Review

18

786-O cells (Supplementary Figure 8D-E). It should be noted, however, that the degree

of target suppression was variable among the different miRNAs in different cell lines.

This can be due to a number of factors including the number of recognition sites (miRNA

response elements) in each target and other cell-specific factors.

We also then examined the effects of miR-192, miR-194 and miR-215 on ZEB2,

MDM2 and TYMS expression at the protein level. MDM2 protein expression was

significantly reduced when CAKI-1 cells were transfected with miR-192, miR-194, or

miR-215 (p < 0.0001, p = 0.0015 and p < 0.0001, respectively). This effect was largely

overcome when co-transfecting the cells with each of these miRNA and its inhibitor

(Figure 4A-B). The protein expression of TYMS was similarly significantly decreased in

cells transfected with miR-192, miR-194 or miR-215 compared to control cells (p =

0.0320, p = 0.0052, and p = 0.0070, respectively). Protein expression levels were

partially restored in cells co-transfected with the miRNA and its inhibitor

(Supplementary figure 9). Our results also show that overexpression of miR-192, miR-

194, or miR-215 significantly reduced ZEB2 protein expression in CAKI-1 cells

compared to control cells, and that expression levels can be partially restored when cells

are co-transfected with each of these miRNAs and its inhibitor (Supplementary Figure

10).

In the second approach, we further validated these miRNA-target interactions by

measuring the change of fluorescence signal of a luciferase vector containing the 3’UTR

of the target upon miRNA transfection. This system provides more evidence about a


123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960



ownloaded from


For Peer Review

19

“direct” interaction between the miRNA and its target. The 786-0 cell line was

transfected with either the Luciferase reporter plasmids containing the 3’UTR of TYMS,

MDM2 or ZEB2, or co-transfected with the plasmids and the miRNA (miR-192, miR-

194, or miR-215). Luciferase activity decreased significantly in cells co-transfected with

plasmids of the target and the targeting miRNA, compared to cells transfected with target

plasmid only (Figure 4C-E).

The third approach was to examine the presence of negative correlation between

miR-192, miR-194 and miR-215 and their predicted targets, MDM2, ZEB2 and TYMS

by qRT-PCR in vivo in patient tissues. We examined the expression levels of the three

miRNAs and their targets on 20 fresh frozen primary ccRCC tissues. As shown in Figure

5, we observed a negative correlation between these three miRNAs and their targets, with

lower expression levels of the miRNA associated with higher expression of its target in

the same patient, and vice versa. This provides indirect evidence that MDM2, ZEB2 and

TYMS are targets of miR-192, miR-194 and miR-215 in vivo.

An interesting observation was the high degree of overlap between targets of all

three miRNAs. This was predicted by target prediction algorithms and experimentally

validated. We further confirmed this overlap be performing sequence alignment of these

miRNAs and the 3'UTR of their predicted targets (MDM2, ZEB2 and TYMS) using the

European Molecular Biology Open Software Suite

(http://www.ebi.ac.uk/Tools/psa/emboss_needle/nucleotide.html). Interestingly, miR-192

and miR-215 not only shared the same seed sequence, but also their mature sequences are


123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960



ownloaded from


For Peer Review

20

almost identical. They only differ in two nucleotides (the 1st and the 20

th nucleotides). In

addition, miR-194 showed partial complementarity with both miRNAs (data not shown).

Also, all three miRNAs show partial complementarity with the 3’UTR of the three

targets. We further validated the target predictions using an independent algorithm (the

online microRNA prediction tool utilizing the PITA algorithm based on sequence

analysis, http://132.77.150.113/pubs/mir07/mir07_prediction.html), as a shown in

Supplementary Table 4.

miR-215 as a prognostic marker for renal cell carcinoma

As miR-215 was significantly differentially expressed between primary and

metastatic ccRCC [17], we hypothesized that this miRNA can serve as a potential

prognostic marker. We examined the expression of miR-215 with the "gold standard"

qRT-PCR using miRNA-specific probes in 61 formalin-fixed paraffin-embedded tissues

of primary ccRCC. Kaplan-Meier survival curve (Figure 6A) showed that lower

expression of miR-215 was associated with significantly decreased disease-free survival

time (Patients with lower miR-215 expression = 26.4 months vs. patients with higher

miR-215 expression = 49.2 months, p=0.0320).

We further validated these data in silico on an independent dataset of 218 primary

ccRCC cases with available overall survival data from The Cancer Genome Atlas and

observed lower expression of miR-215 to be associated with significantly worse survival (

p = 0.0032) (Figure 6B).


123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960



ownloaded from


For Peer Review

21

miR-192, miR-194 and miR-215 are conserved among species

We used the University of California Santa Cruise (UCSC) Genome Browser for

sequence comparison of miR-192, miR-194 and miR-215 among species. Our analysis

shows the high conservation of these three miRNAs among 28 species (Supplementary

Figure 11). Conservation among species indicates that these miRNAs may have vital

functions that are maintained during evolution.


123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960



ownloaded from


For Peer Review

22

Discussion

In our previous work, we identified a miRNA signature of metastatic ccRCC.

miR-192, miR-194 and miR-215 were significantly differentially expressed in metastatic,

compared to primary ccRCC. In the current study, we provide evidence that these three

miRNAs can be involved in RCC progression and that miR-215 is a potential prognostic

marker for ccRCC. We also identified and validated three of their target genes; ZEB2,

MDM2 and TYMS.

miR-192, miR-194 and miR-215 are highly enriched in the normal kidney [18],

indicating that they play a role in kidney development and differentiation. In our previous

work, we reported that these three miRNAs are significantly downregulated in primary

ccRCC compared to normal kidney tissue [27]. These three miRNAs are further

downregulated in metastatic ccRCC [17]. Taken together, this step-wise downregulation

indicates their involvement in controlling tumor-suppressor pathways. This is also

supported by the fact that these three miRNAs are present in two clusters. Members of

the same cluster are usually co-expressed and co-regulated. The miR-215 / miR-194-1

cluster is located within the common fragile site FRA1H (1q41~q42.1) which is deleted

in many cancers [28]. 1q41 was reported to be associated with breast and esophageal

cancer metastasis. The downregulation of these three miRNAs was also reported in other

cancers including nephroblastoma, myeloma, colon, and gastric cancers [20;21].

Our results show that the main effect of these three miRNAs is on tumor

migration and invasion. Little effect was shown on tumor proliferation, although this was


123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960



ownloaded from


For Peer Review

23

not statistically significant. This is not unprecedented. Recent literature showing that the

key contribution of some of these miRNAs is on tumor invasion and migration abilities,

which are essential features for metastasis and EMT [29;30]. Moreover, our results

should be interpreted with caution as it might be cell or tissue type specific. Others have

also shown more significant effect on cell proliferation in other cell types”.

Our in silico analysis showed that miR-192, miR-194 and miR-215 can target

ZEB2, MDM2 and TYMS. MDM2 is a key inhibitor of p53. It activates HIF-1α and

VEGF activity (23). MDM2 overexpression is reported to be associated with metastasis

in many cancers. Its overexpression decreases E-cadherin levels with subsequent increase

in cell motility in breast carcinoma (24). It was also reported to increase cell motility and

invasiveness in RCC (25). TYMS is essential for DNA synthesis and its inhibition is

reported to block DNA replication and repair (26). TYMS SNP variations are associated

with increased risk of RCC (27). TYMS upregulation was reported in RCC and correlates

with tumor progression (28). It is also a target of the 5-fluorouracil (5-FU) used in

advanced RCC. It was also shown to have a prognostic significance in bladder cancer.

SIP1/ZEB2 represses E-cadherin and it was shown to be involved in epithelial to

mesenchymal transition (EMT) which is a key process in tumor progression. ZEB2 can

mediate the HIF-1 repression effect of E-cadherin in RCC.

We experimentally validated our in silico predictions using independent

approaches. These results, however, should be interpreted with caution, due to the

possibility of indirect targeting and off-target effects. The luciferase system can provide


123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960



ownloaded from


For Peer Review

24

more evidence of direct miRNA-target interactions. Finally, we provide in vivo evidence

by documenting the negative correlation between the expression of these three miRNAs

and these targets in ccRCC patients' tissues. Our findings are also consistent with recent

reports showing that miR-192 and miR-215 can target TYMS in colon cancer [31].

MDM2 is also a target of these miRNAs in myeloma [31].

Interestingly, these miRNAs occur in two clusters. Members of the same cluster

can have coordinated effects. They may target the same molecule (convergent targeting)

or may hit several molecules in the same or related biological pathways (divergent

targeting), as demonstrated in recent reports [32;33]. The remarkable target overlap can

be explained by the fact that miR-192 and miR-215 have similar seed sequence which is

highly important for miRNA target interaction. It should be also noted that 3’

supplementary sites can enhance the seed pairing. Pairing to the 3׳ region includes mainly

the nucleotides 13-16. Furthermore, this 3׳ pairing can also compensate for nucleotide

mismatch in the seed region [34]. miRNAs were reported to target not only the 3׳UTR

but also 5׳UTR, coding regions, promoters and gene termini. Our results are not

unprecedented; Senanayake et al identified ACVR2B to be a common target for miR-

192, miR-194 and miR-215 in renal childhood neoplasms [35].

The involvement of these miRNAs in tumor progression is not surprising and is

supported by previous reports. miR-192, miR-194 and miR-215 are induced by p53 and

also they were reported to be p53 positive regulators [20;21]. Kim et al. demonstrated

that p53 can regulate epithelial-mesenchymal transition through targeting ZEB2 by miR-


123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960



ownloaded from


For Peer Review

25

192 family. Also, Krishnamachary et al. showed that E-cadherin repression by HIF-1 can

be mediated by ZEB2 in RCC [36]. Also, MDM2 overexpression correlates with tumor

progression and metastasis in different cancers, including RCC [37-41].

Our findings can also have therapeutic implications. TYMS is a target of the 5-FU

anticancer agents which is now being considered in combination therapies for advanced

RCC. Recently, using RCC xenograft model, 5-FU was shown to enhance the Sorafenib

and Sunitinib antitumor effect [42]. Recently, MDM2 is being investigated as a potential

new therapeutic target in colon cancer [43]. Also, Vastsyayan et al demonstrated that

MDM2 inhibitor (Nutlin-3) can enhance the effect of Sorafenib in RCC [44].

The fact that one miRNA can hit multiple targets attracted the attention to the

miRNAs as promising therapies in cancer. The different strategies for using miRNA

therapy were recently reviewed including miRNA replacement using adeno-associated

viral vectors and synthetic miRNA precursors in hepatocellular carcinoma and prostate

cancer respectively [45] . Also, targeting miRNAs using synthetic molecules can be of

therapeutic interest.

In conclusion, we showed that miR-192, miR-194 and miR-215 have tumor

suppressor effects on RCC by reducing the cellular migration and invasion abilities. We

identified potential mechanisms through which these three miRNAs can negatively affect

these biological processes by targeting key molecules involved in metastasis. These three

miRNAs can have hundreds of targets and a global analysis of the overall spectrum of


123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960



ownloaded from


For Peer Review

26

changes of miRNAs would be more suitable. However, due to limited resources we did a

targeted approach to select the targets that are of clinical significance that was shown in

other cancers. This however does not exclude the presence of other significant targets that

are yet to be identified and validated. This is a net effect of miRNAs and there might be a

number of underlined, direct and indirect, and sometimes being even opposing target

effects. Also, we demonstrated that miR-215 can be potential prognostic marker in RCC.

Our results can be a step forward towards developing a new therapeutic target in RCC.


123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960



ownloaded from


For Peer Review

27

Supplementary Material

Supplementary figures 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 & 11 and Supplementary tables 1, 2, 3 &

4 can be found at http://carcin.oxfordjournals.org/

Funding

This work was supported by grants from the Canadian Institute of Health Research

(CIHR grant # 119606), Canadian Cancer Society (CCS grant # 20185), and the Kidney

Foundation of Canada.

Conflict of Interest

The authors declared no conflict of interest


123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960



ownloaded from


For Peer Review

28

Reference List

1. Chow, W.H.et al., (2010) Epidemiology and risk factors for kidney cancer.

Nat.Rev.Urol., 7, 245-257.

2. Falebita, O.A.et al., (2009) Rising incidence of renal cell carcinoma in Ireland.

Int.Urol.Nephrol., 41, 7-12.

3. Chaffer, C.L.et al., (3-25-2011) A perspective on cancer cell metastasis. Science,

331, 1559-1564.

4. Zhang, R.et al., (2009) Small but influential: the role of microRNAs on gene

regulatory network and 3'UTR evolution. J.Genet.Genomics, 36, 1-6.

5. Sotiropoulou, G.et al., (2009) Emerging roles of microRNAs as molecular switches

in the integrated circuit of the cancer cell. RNA., 15, 1443-1461.

6. White, N.M.et al., (2011) Metastamirs: a stepping stone towards improved cancer

management. Nat.Rev.Clin.Oncol., 8, 75-84.

7. Jung, M.et al., (2009) MicroRNA profiling of clear cell renal cell cancer identifies a

robust signature to define renal malignancy. J.Cell Mol.Med., 13, 3918-3928.

8. White, N.M.et al., (2011) miRNA profiling for clear cell renal cell carcinoma:

biomarker discovery and identification of potential controls and consequences of

miRNA dysregulation. J.Urol., 186, 1077-1083.

9. Chow, T.F.et al., (2010) The miR-17-92 cluster is over expressed in and has an

oncogenic effect on renal cell carcinoma. J.Urol., 183, 743-751.

10. Lichner, Z.et al., (2012) Pleiotropic action of renal cell carcinoma-dysregulated

miRNAs on hypoxia-related signaling pathways. Am.J.Pathol., 180, 1675-1687.

11. Redova, M.et al., (2-11-2011) MicroRNAs and their target gene networks in renal

cell carcinoma. Biochem.Biophys.Res.Commun., 405, 153-156.

12. White, N.M.et al., (2010) MicroRNAs: exploring a new dimension in the

pathogenesis of kidney cancer. BMC.Med., 8, 65.

13. Metias, S.M.et al., (2009) MicroRNAs in clinical oncology: at the crossroads

between promises and problems. J.Clin.Pathol., 62, 771-776.

14. White, N.M.et al., (2011) Translating molecular signatures of renal cell carcinoma

into clinical practice. J.Urol., 186, 9-11.


123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960



ownloaded from


For Peer Review

29

15. Heinzelmann, J.et al., (2011) Specific miRNA signatures are associated with

metastasis and poor prognosis in clear cell renal cell carcinoma. World J.Urol., 29,

367-373.

16. Khella, H.W.et al., (2012) Exploring the role of miRNAs in renal cell carcinoma

progression and metastasis through bioinformatic and experimental analyses.

Tumour.Biol., 33, 131-140.

17. White, N.M.et al., (11-22-2011) miRNA profiling in metastatic renal cell carcinoma

reveals a tumour-suppressor effect for miR-215. Br.J.Cancer, 105, 1741-1749.

18. Sun, Y.et al., (2004) Development of a micro-array to detect human and mouse

microRNAs and characterization of expression in human organs. Nucleic Acids

Res., 32, e188.

19. Saal, S.et al., (2009) MicroRNAs and the kidney: coming of age.

Curr.Opin.Nephrol.Hypertens., 18, 317-323.

20. Braun, C.J.et al., (12-15-2008) p53-Responsive micrornas 192 and 215 are capable

of inducing cell cycle arrest. Cancer Res., 68, 10094-10104.

21. Pichiorri, F.et al., (10-19-2010) Downregulation of p53-inducible microRNAs 192,

194, and 215 impairs the p53/MDM2 autoregulatory loop in multiple myeloma

development. Cancer Cell, 18, 367-381.

22. Dong, P.et al., (2011) MicroRNA-194 inhibits epithelial to mesenchymal transition

of endometrial cancer cells by targeting oncogene BMI-1. Mol.Cancer, 10, 99.

23. Wotschofsky, Z.et al., (10-15-2011) Reference genes for the relative quantification

of microRNAs in renal cell carcinomas and their metastases. Anal.Biochem., 417,

233-241.

24. White, N.M.et al., (4-13-2010) Three dysregulated miRNAs control kallikrein 10

expression and cell proliferation in ovarian cancer. Br.J.Cancer, 102, 1244-1253.

25. White, N.M.et al., (2010) Dysregulation of kallikrein-related peptidases in renal cell

carcinoma: potential targets of miRNAs. Biol.Chem., 391, 411-423.

26. Camp, R.L.et al., (11-1-2004) X-tile: a new bio-informatics tool for biomarker

assessment and outcome-based cut-point optimization. Clin.Cancer Res., 10, 7252-

7259.

27. White, N.M.et al., (2011) miRNA profiling for clear cell renal cell carcinoma:

biomarker discovery and identification of potential controls and consequences of

miRNA dysregulation. J.Urol., 186, 1077-1083.


123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960



ownloaded from


For Peer Review

30

28. Curatolo, A.et al., (2007) Molecular characterization of the human common fragile

site FRA1H. Genes Chromosomes.Cancer, 46, 487-493.

29. Le, X.F.et al., (2012) Modulation of MicroRNA-194 and cell migration by HER2-

targeting trastuzumab in breast cancer. PLoS.One., 7, e41170.

30. Meng, Z.et al., (2010) miR-194 is a marker of hepatic epithelial cells and

suppresses metastasis of liver cancer cells in mice. Hepatology, 52, 2148-2157.

31. Boni, V.et al., (2010) miR-192/miR-215 influence 5-fluorouracil resistance through

cell cycle-mediated mechanisms complementary to its post-transcriptional

thymidilate synthase regulation. Mol.Cancer Ther., 9, 2265-2275.

32. Wang, J.et al., (2011) Regulatory coordination of clustered microRNAs based on

microRNA-transcription factor regulatory network. BMC.Syst.Biol., 5, 199.

33. Yuan, X.et al., (2009) Clustered microRNAs' coordination in regulating protein-

protein interaction network. BMC.Syst.Biol., 3, 65.

34. Bartel, D.P.et al., (1-23-2009) MicroRNAs: target recognition and regulatory

functions. Cell, 136, 215-233.

35. Senanayake, U.et al., (2012) miR-192, miR-194, miR-215, miR-200c and miR-141

are downregulated and their common target ACVR2B is strongly expressed in renal

childhood neoplasms. Carcinogenesis, 33, 1014-1021.

36. Krishnamachary, B.et al., (3-1-2006) Hypoxia-inducible factor-1-dependent

repression of E-cadherin in von Hippel-Lindau tumor suppressor-null renal cell

carcinoma mediated by TCF3, ZFHX1A, and ZFHX1B. Cancer Res., 66, 2725-

2731.

37. Bahnassy, A.A.et al., (2012) Aberrant expression of cell cycle regulatory genes

predicts overall and disease free survival in malignant pleural mesothelioma

patients. Exp.Mol.Pathol., 93, 154-161.

38. Haitel, A.et al., (2000) mdm2 expression as a prognostic indicator in clear cell renal

cell carcinoma: comparison with p53 overexpression and clinicopathological

parameters. Clin.Cancer Res., 6, 1840-1844.

39. Hirata, H.et al., (7-15-2007) MDM2 SNP309 polymorphism as risk factor for

susceptibility and poor prognosis in renal cell carcinoma. Clin.Cancer Res., 13,

4123-4129.

40. Noon, A.P.et al., (2-15-2010) p53 and MDM2 in renal cell carcinoma: biomarkers

for disease progression and future therapeutic targets?. Cancer, 116, 780-790.


123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960



ownloaded from


For Peer Review

31

41. Noon, A.P.et al., (2012) Combined p53 and MDM2 biomarker analysis shows a

unique pattern of expression associated with poor prognosis in patients with renal

cell carcinoma undergoing radical nephrectomy. BJU.Int., 109, 1250-1257.

42. Miyake, M.et al., (2012) 5-fluorouracil enhances the antitumor effect of sorafenib

and sunitinib in a xenograft model of human renal cell carcinoma. Oncol.Lett., 3,

1195-1202.

43. Hav, M.et al., (2011) MDM2 gene amplification and protein expressions in colon

carcinoma: is targeting MDM2 a new therapeutic option?. Virchows Arch., 458,

197-203.

44. Vatsyayan, R.et al., (10-17-2011) Nutlin-3 enhances sorafenib efficacy in renal cell

carcinoma. Mol.Carcinog..

45. Gandellini, P.et al., (2011) MicroRNAs as new therapeutic targets and tools in

cancer. Expert.Opin.Ther.Targets., 15, 265-279.


123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960



ownloaded from


For Peer Review

32

Figure Legends

Fig. 1. Overexpression of miR-192, miR-194, or miR-215 has negative effect on

cellular migration. (A) Representative photomicrographs showing the effect of miR-192

expression on the migration rate of the 786-O RCC cell line. The top row shows the cells

at the time of wounding (0 h), and the bottom row shows cellular migration after 9 hours.

Overexpression of miR-192 led to significant reduction in the rate of cell migration, with

incomplete wound closure after 9h, compared to controls. This was restored when co-

transfecting miR-192 and its inhibitor. (B) Representative bar graph showing the effect of

miR-192 on cell migration. Comparable results were obtained for miR-194 (C), and miR-

215 (D).

Fig. 2. miR-192, miR-194 and miR-215 have negative effect on cellular invasion. (A)

Representative photomicrographs showing the effect of miR-194 on cell migration in

786-O RCC cell line by comparing the number of cells invaded through the matrigel

inserts to the number of cells invaded through the control inserts. Control inserts are

shown in the top panel and matrigel inserts in the lower panel. miR-194 transfection

resulted in significant reduction of cell invasion ability compared to untransfected cells

and cells transfected with transfection agent only, scrambled miRNA (negative control),

and anti-miR-194. Partial restoration of cell invasion was obtained when co-transfection

with miR-194 and its inhibitor. (B) A representative bar graph showing the effect of miR-

194 on cell invasion. Comparable results were obtained for miR-192 (C) and miR-215

(D).


123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960



ownloaded from


For Peer Review

33

Fig. 3. miR-192, miR-194 and miR-215 can target ZEB2. Representative bar graphs

showing that ZEB2 expression was significantly decreased, at the mRNA level, in 786-O

cells, upon transfection of miR-192 (A), miR-194 (B), and miR-215 (C). These effects

were partially restored upon the co-transfection of the miRNA and its inhibitor.

Expressions are shown as relative expression values compared to control untrasnfected

cells to the far left. NC; negative control of scrambled miRNA sequences.

Fig. 4. (Fig. 4A-B) MDM2 is a target of miR-192, miR-194 and miR-215. (A)

Representative Western blot analysis showing decreased MDM2 protein level in CAKI-1

cells transfected with miR-192, miR-194 or miR-215 compared to cells transfected with

the transfection agent only or co-transfected with the miRNA and its inhibitor. (B) Bar

graph showing the quantification of changes in protein expression levels in the gel.

Expression values were compared to control untransfected cells. α tubulin was used as a

loading control. (Fig. 4C-E). MDM2, ZEB2 and TYMS are targets of miR-192, miR-

194 and miR-215. Co-transfection of 786-0 cells with reporter vectors containing the

3’UTR of MDM2 (C), ZEB2 (D), or TYMS (E) and miR-192, miR-194, or miR-215

significantly decreased luciferase activity compared to cells transfected with the

constructs only.

Fig. 5. A negative correlation was observed between the expression levels of miR-192

and miR-215 and their predicted targets, ZEB2 and MDM2 in patient tissues. The

graphs show pair-wise comparison between each of the miRNAs and its target for each

patient. Our results show the presence of a negative correlation between these miRNAs


123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960



ownloaded from


For Peer Review

34

and their predicted targets with lower expression of miR-192 and miR-215 associated

with higher expression of MDM2 (A and B) and ZEB2 (C and D), and vice versa. This

provides indirect evidence that ZEB2 and MDM2 are targets of miR-192 and miR-215.

Patient cases are shown on the X axes and the relative expression values are presented

along the Y axes. Expression levels are shown as relative expression values of the

miRNAs and their targets normalized against an internal control in the same specimen.

Fig. 6. (A) Kaplan-Meier disease-free survival (DFS) plot comparing miR-215

expression in RCC patients. miR-215 expression was dichotomized into high and low

expression categories. Patients with lower miR-215 expression had significantly lower

disease-free survival compared to those with high expression (p = 0.032). (B) Kaplan-

Meier overall survival (OS) plot comparing miR-215 expression in RCC patients.

‘Level 3’ miRNA expression data (normalized gene expression generated using the

Illumina GA miRNASeq platform) for miR-215 in ccRCC and overall survival data were

obtained from The Cancer Genome Atlas (TCGA). Lower expression of miR-215 is

significantly associated with worse survival (p = 0.0032).

Supplementary Fig. 1. The 786-O, ACHN and CAKI-1 kidney cancer cell lines have

low endogenous expression levels of miR-192, miR-194 and miR-215. The expression

levels of miR-192, miR-194 and miR-215 in 786-O, ACHN and CAKI-1 RCC cell lines

were measured by qRT-PCR. Expression values are shown as relative expression

compared to a pool of patient primary ccRCC tumor tissues. All three cell lines have very

low endogenous expression levels of miR-192, miR-194 and miR-215.


123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960



ownloaded from


For Peer Review

35

Supplementary Fig. 2. Confirmation of successful transfection of miR-194 in CAKI-

1 cells. A time-course experiment showing a significant increase in miR-194 expression

(measured by qRT-PCR) after transfection. Higher expression levels were maintained for

3 days.

Supplementary Fig. 3. miR-192, miR-194 and miR-215 have negative effect on

cellular invasion. A representative bar graph showing the effect of these three miRNAs

in the ACHN cells. Cells transfected with either of miR-192, miR-194, or miR-215

showed significantly decreased cellular invasion compared to the control cells. This

effect was partially restored by co-transfection of the miRNA and its inhibitor.

Supplementary Fig. 4. miR-192, miR-194 and miR-215 overexpression have

insignificant effect on cellular proliferation. ACHN cells were transfected with miR-

192, miR-194, or miR-215. This resulted in a slight insignificant reduction in the rate of

cell proliferation, compared to controls.

Supplementary Fig. 5. 786-O, ACHN and CAKI-1 cells have high endogenous

expression levels of MDM2, ZEB2 and TYMS. The endogenous expression levels of

MDM2, ZEB2 and TYMS in 786-O, ACHN and CAKI-1 RCC cell lines was measured

using qRT-PCR. All three cell lines have high endogenous expression levels of MDM2,

ZEB2 and TYMS. Expression is shown as arbitrary units normalized against endogenous

control. P = a pool of primary ccRCC tissues; M = pool of metastatic ccRCC tissues


123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960



ownloaded from


For Peer Review

36

Supplementary Fig. 6. miR-192, miR-194 and miR-215 can target ZEB2. (A)

Representative bar graphs showing that ZEB2 expression was significantly decreased, at

the mRNA level, in CAKI-1 cells, upon transfection of miR-194. (B) A representative

graph showing the ability of miR-192 overexpression to reduce ZEB2 expression in

ACHN kidney cancer cells. These effects were partially restored upon the co-transfection

of the miRNA and its inhibitor. Expressions are shown as relative expression values

compared to control untrasnfected cells to the far left. NC; negative control of scrambled

miRNA sequences.

Supplementary Fig. 7. miR-192, miR-194 and miR-215 can target TYMS at the

mRNA level. (A-C) Representative graphs showing that TYMS expression was

decreased in CAKI-1 cells upon overexpression of each of these miRNAs. (D) A

representative graph showing a significant reduction of TYMS in 786-O cells upon miR-

194 overexpression. (E) A representative graph in ACHN kidney cancer cells showing

the ability of miR-192 to significantly reduce TYMS mRNA expression. These effects

were partially restored upon the con-transfection of the miRNA and its inhibitor.

Expressions are shown as relative expression values compared to control cells to the far

left. NC; negative control of scrambled miRNA sequences.

Supplementary Fig. 8. miR-192, miR-194 and miR-215 can target MDM2 at the

mRNA level. (A-C) MDM2 expression was decreased in ACHN cells upon


123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960



ownloaded from


For Peer Review

37

overexpression of each of the three miRNAs. (D) A representative graph in CAKI-1 cells

showing the ability of miR-194 to reduce MDM2 expression. (E) A representative graph

in 786-O cells showing that miR-215 overexpression resulted in significant reduction of

MDM2 expression. Expressions are shown as relative expression values compared to

control cells to the far left.

Supplementary Fig. 9. TYMS is a target of miR-192, miR-194 and miR-215. (A)

Representative Western blot analysis showing decreased TYMS protein level in CAKI-1


the transfection agent only or co-transfected with the corresponding miRNA and its

inhibitor. (B) Bar graph showing the quantification of changes in protein expression

levels in the gel. Expression values were compared to control untransfected cells.

Supplementary Fig. 10. ZEB2 is a target of miR-192, miR-194 and miR-215. (A)

Representative Western blot analysis showing decreased ZEB2 protein level in CAKI-1


the transfection agent only. Proteins levels were partially restored when co-transfecting

the cells with the miRNA and its inhibitor. (B) Bar graph showing the quantification of

changes in protein expression levels in the gel. Expression values were compared to

control untransfected cells.


123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960



ownloaded from


For Peer Review

38

Supplementary Fig. 11 (A-B). miR-192, miR-194 and miR-215 are highly conserved

among different species. Data were analyzed according to the University of California

Santa Cruz Human Genome browser.


123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960



ownloaded from


For Peer Review

Fig. 1. Overexpression of miR-192, miR-194, or miR-215 has negative effect on cellular migration. (A) Representative photomicrographs showing the effect of miR-192 expression on the migration rate of the 786-O RCC cell line. The top row shows the cells at the time of wounding (0 h), and the bottom row shows

cellular migration after 9 hours. Overexpression of miR-192 led to significant reduction in the rate of cell migration, with incomplete wound closure after 9h, compared to controls. This was restored when co-

transfecting miR-192 and its inhibitor. 182x76mm (300 x 300 DPI)


123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960



ownloaded from


For Peer Review

Fig. 1. Overexpression of miR-192, miR-194, or miR-215 has negative effect on cellular migration.(B) Representative bar graph showing the effect of miR-192 on cell migration. Comparable results were

obtained for miR-194 (C), and miR-215 (D).

65x22mm (300 x 300 DPI)


123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960



ownloaded from


For Peer Review

Fig. 2. miR-192, miR-194 and miR-215 have negative effect on cellular invasion. (A) Representative photomicrographs showing the effect of miR-194 on cell migration in 786-O RCC cell line by comparing the number of cells invaded through the matrigel inserts to the number of cells invaded through the control

inserts. Control inserts are shown in the top panel and matrigel inserts in the lower panel. miR-194 transfection resulted in significant reduction of cell invasion ability compared to untransfected cells and cells transfected with transfection agent only, scrambled miRNA (negative control), and anti-miR-194. Partial

restoration of cell invasion was obtained when co-transfection with miR-194 and its inhibitor. 182x78mm (300 x 300 DPI)


123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960



ownloaded from


For Peer Review

Fig. 2. miR-192, miR-194 and miR-215 have negative effect on cellular invasion. (B) A representative bar graph showing the effect of miR-194 on cell invasion. Comparable results were obtained for miR-192 (C)

and miR-215 (D).

65x22mm (300 x 300 DPI)


123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960



ownloaded from


For Peer Review

Fig. 3. miR-192, miR-194 and miR-215 can target ZEB2. Representative bar graphs showing that ZEB2 expression was significantly decreased, at the mRNA level, in 786-O cells, upon transfection of miR-192 (A), miR-194 (B), and miR-215 (C). These effects were partially restored upon the co-transfection of the miRNA

and its inhibitor. Expressions are shown as relative expression values compared to control untrasnfected cells to the far left. NC; negative control of scrambled miRNA sequences.

77x32mm (300 x 300 DPI)


123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960



ownloaded from


For Peer Review

Fig. 4. (Fig. 4A-B) MDM2 is a target of miR-192, miR-194 and miR-215. (A) Representative Western blot analysis showing decreased MDM2 protein level in CAKI-1 cells transfected with miR-192, miR-194 or miR-215 compared to cells transfected with the transfection agent only or co-transfected with the miRNA and its

inhibitor. (B) Bar graph showing the quantification of changes in protein expression levels in the gel. Expression values were compared to control untransfected cells. α tubulin was used as a loading control.

104x59mm (300 x 300 DPI)


123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960



ownloaded from


For Peer Review

Fig. 4. (Fig. 4C-E). MDM2, ZEB2 and TYMS are targets of miR-192, miR-194 and miR-215. Co-transfection of 786-0 cells with reporter vectors containing the 3’UTR of MDM2 (C), ZEB2 (D), or TYMS (E) and miR-192, miR-194, or miR-215 significantly decreased luciferase activity compared to cells transfected with the

constructs only. 129x90mm (300 x 300 DPI)


123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960



ownloaded from


For Peer Review

Fig. 5. A negative correlation was observed between the expression levels of miR-192 and miR-215 and their predicted targets, ZEB2 and MDM2 in patient tissues. The graphs show pair-wise comparison between each of the miRNAs and its target for each patient. Our results show the presence of a negative correlation

between these miRNAs and their predicted targets with lower expression of miR-192 and miR-215 associated with higher expression of MDM2 (A and B) and ZEB2 (C and D), and vice versa. This provides

indirect evidence that ZEB2 and MDM2 are targets of miR-192 and miR-215. Patient cases are shown on the X axes and the relative expression values are presented along the Y axes. Expression levels are shown as relative expression values of the miRNAs and their targets normalized against an internal control in the

same specimen. 104x58mm (300 x 300 DPI)


123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960



ownloaded from


For Peer Review

Fig. 6. (A) Kaplan-Meier disease-free survival (DFS) plot comparing miR-215 expression in RCC patients. miR-215 expression was dichotomized into high and low expression categories. Patients with lower miR-215 expression had significantly lower disease-free survival compared to those with high expression (p = 0.032).

(B) Kaplan-Meier overall survival (OS) plot comparing miR-215 expression in RCC patients. ‘Level 3’ miRNA expression data (normalized gene expression generated using the Illumina GA miRNASeq platform) for miR-

215 in ccRCC and overall survival data were obtained from The Cancer Genome Atlas (TCGA). Lower expression of miR-215 is significantly associated with worse survival (p = 0.0032).

69x26mm (300 x 300 DPI)


123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960



ownloaded from


For Peer Review

Supplementary Fig. 1. The 786-O, ACHN and CAKI-1 kidney cancer cell lines have low endogenous expression levels of miR-192, miR-194 and miR-215. The expression levels of miR-192, miR-194 and miR-215 in 786-O, ACHN and CAKI-1 RCC cell lines were measured by qRT-PCR. Expression values are shown as

relative expression compared to a pool of patient primary ccRCC tumor tissues. All three cell lines have very low endogenous expression levels of miR-192, miR-194 and miR-215.

106x61mm (300 x 300 DPI)


123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960



ownloaded from


For Peer Review

Supplementary Fig. 2. Confirmation of successful transfection of miR-194 in CAKI-1 cells. A time-course experiment showing a significant increase in miR-194 expression (measured by qRT-PCR) after transfection.

Higher expression levels were maintained for 3 days.

156x132mm (300 x 300 DPI)


123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960



ownloaded from


For Peer Review

Supplementary Fig. 3. miR-192, miR-194 and miR-215 have negative effect on cellular invasion. A representative bar graph showing the effect of these three miRNAs in the ACHN cells. Cells transfected with either of miR-192, miR-194, or miR-215 showed significantly decreased cellular invasion compared to the

control cells. This effect was partially restored by co-transfection of the miRNA and its inhibitor. 165x150mm (300 x 300 DPI)


123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960



ownloaded from


For Peer Review

Supplementary Fig. 4. miR-192, miR-194 and miR-215 overexpression have insignificant effect on cellular proliferation. ACHN cells were transfected with miR-192, miR-194, or miR-215. This resulted in a slight

insignificant reduction in the rate of cell proliferation, compared to controls. 99x53mm (300 x 300 DPI)


123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960



ownloaded from


For Peer Review

Supplementary Fig. 5. 786-O, ACHN and CAKI-1 cells have high endogenous expression levels of MDM2, ZEB2 and TYMS. The endogenous expression levels of MDM2, ZEB2 and TYMS in 786-O, ACHN and CAKI-1 RCC cell lines was measured using qRT-PCR. All three cell lines have high endogenous expression levels of MDM2, ZEB2 and TYMS. Expression is shown as arbitrary units normalized against endogenous control. P =

a pool of primary ccRCC tissues; M = pool of metastatic ccRCC tissues 183x115mm (300 x 300 DPI)


123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960



ownloaded from


For Peer Review

Supplementary Fig. 6. miR-192, miR-194 and miR-215 can target ZEB2. (A) Representative bar graphs showing that ZEB2 expression was significantly decreased, at the mRNA level, in CAKI-1 cells, upon

transfection of miR-194. (B) A representative graph showing the ability of miR-192 overexpression to reduce ZEB2 expression in ACHN kidney cancer cells. These effects were partially restored upon the co-transfection of the miRNA and its inhibitor. Expressions are shown as relative expression values compared to control

untrasnfected cells to the far left. NC; negative control of scrambled miRNA sequences. 91x46mm (300 x 300 DPI)


123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960



ownloaded from


For Peer Review

Supplementary Fig. 7. miR-192, miR-194 and miR-215 can target TYMS at the mRNA level. (A-C) Representative graphs showing that TYMS expression was decreased in CAKI-1 cells upon overexpression of

each of these miRNAs.

80x35mm (300 x 300 DPI)


123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960



ownloaded from


For Peer Review

Supplementary Fig. 7. miR-192, miR-194 and miR-215 can target TYMS at the mRNA level. (D) A representative graph showing a significant reduction of TYMS in 786-O cells upon miR-194 overexpression. (E) A representative graph in ACHN kidney cancer cells showing the ability of miR-192 to significantly reduce TYMS mRNA expression. These effects were partially restored upon the con-transfection of the miRNA and its inhibitor. Expressions are shown as relative expression values compared to control cells to the far left.

NC; negative control of scrambled miRNA sequences. 98x53mm (300 x 300 DPI)


123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960



ownloaded from


For Peer Review

Supplementary Fig. 8. miR-192, miR-194 and miR-215 can target MDM2 at the mRNA level. (A-C) MDM2 expression was decreased in ACHN cells upon overexpression of each of the three miRNAs.

82x36mm (300 x 300 DPI)


123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960



ownloaded from


For Peer Review

Supplementary Fig. 8. miR-192, miR-194 and miR-215 can target MDM2 at the mRNA level. (D) A representative graph in CAKI-1 cells showing the ability of miR-194 to reduce MDM2 expression. (E) A

representative graph in 786-O cells showing that miR-215 overexpression resulted in significant reduction of MDM2 expression. Expressions are shown as relative expression values compared to control cells to the far

left. 98x53mm (300 x 300 DPI)


123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960



ownloaded from


For Peer Review

Supplementary Fig. 9. TYMS is a target of miR-192, miR-194 and miR-215. (A) Representative Western blot analysis showing decreased TYMS protein level in CAKI-1 cells transfected with miR-192, miR-194 or miR-215 compared to cells transfected with the transfection agent only or co-transfected with the corresponding

miRNA and its inhibitor. (B) Bar graph showing the quantification of changes in protein expression levels in the gel. Expression values were compared to control untransfected cells.

100x54mm (300 x 300 DPI)


123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960



ownloaded from


For Peer Review

Supplementary Fig. 10. ZEB2 is a target of miR-192, miR-194 and miR-215. (A) Representative Western blot analysis showing decreased ZEB2 protein level in CAKI-1 cells transfected with miR-192, miR-194 or miR-215 compared to cells transfected with the transfection agent only. Proteins levels were partially

restored when co-transfecting the cells with the miRNA and its inhibitor. (B) Bar graph showing the quantification of changes in protein expression levels in the gel. Expression values were compared to control

untransfected cells. 102x56mm (300 x 300 DPI)


123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960



ownloaded from


For Peer Review

Supplementary Fig. 11 A. miR-192 and miR-194 are highly conserved among different species. Data were analyzed according to the University of California Santa Cruz Human Genome browser.

182x82mm (300 x 300 DPI)


123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960



ownloaded from


For Peer Review

Supplementary Fig. 11B. miR-215 and miR-194 are highly conserved among different species. Data were analyzed according to the University of California Santa Cruz Human Genome browser.

182x79mm (300 x 300 DPI)


123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960



ownloaded from


For Peer Review

Supplementary Table (1-A): Thermal cycling conditions for miRNA-specific reverse

transcription using the TaqMan® MicroRNA Reverse Transcription Kit (Applied Biosystems,

Foster City, CA)

Step type Time ( minutes) Temperature (°C)

Hold 30 16

Hold 30 42

Hold 5 85

Hold ∞ 4

Supplementary Table (1-B): Thermal cycling conditions for qRT-PCR using the TaqMan

microRNA Assay® Kit on the Step One™ Plus Real-Time PCR System (Applied Biosystems,

Foster City, CA).

Supplementary Table (1-C): Thermal cycling conditions for reverse transcription using the

High capacity RNA-to-cDNA kit (Applied Biosystems, Foster City, CA)

Step 1 Step 2 Step 3 Step 4

Temperature 25°C 37°C 85°C 4°C

Time 10 min 120 min 5 min ∞

Supplementary Table (1-D): Thermal cycling conditions for qRT-PCR using the Fast Syber

Green Master Mix (Applied Biosystems, Foster City, CA)

Holding stage Cycling stage Melt Curve Stage

Step 1 Step 2 Step 3 Step 1 Step 2 Step 3

Temperature 95°C 95°C 57°C 60°C 95°C 60°C 95°C

Time 20 sec. 5 sec. 20 sec. 30 sec. 15 sec. 1 min. 15 sec

Step type Time ( seconds) Temperature (°C)

Hold 20 95

Cycling 1 95

Cycling 20 60


123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960



ownloaded from


For Peer Review

Supplementary Table (2): The Primer Sequences

Target Forward-Primer Reverse-Primer

ZEB2 5’-TCTCCCATTCTGGTTCCTACA-3’ 5’-TTCTCATTCGGCCATTTACAG-3’

TYMS 5’-ACACTTTGGGAGATGCACATA-3’ 5’-AATAGTTGGATGCGGATTGTA-3’

MDM2 5’-GCCTGGCTCTGTGTGTAATAAG-3’ 5’-TGAGAGTTCTTGTCCTTCTTCACTAA-3’

PPIA 5’-ATGCTGGCCCCAACACAA-3’ 5’-CCCTCTTTCACCTTGCCAAA-3’

HPRT1 5’-TTGCTGACCTGCTGGATTAC-3’ 5’-TCTCCACCAATTACTTTTATGTCC-3’


123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960



ownloaded from


For Peer Review

Supplementary Table (3): miR-192, miR-194 and miR-215 target pathways and key

molecules involved in tumor progression

Pathway miRNA Gene Name

Renal Cell Carcinoma miR-192 ARNT2

miR-215 RAP1B

miR-194 HIF1A, MAPK1, RAC1, PAK2, RAP1B,

AKT2, CRK

VEGF signaling pathway miR-192 NFAT5

miR-215 NFAT5

miR-194 MAPK1, RAC1, AKT2, PRKCB1, NFAT5

TGF-Beta signaling Pathway miR-192 ACVR2A, NOG, ACVR2B, BMPR2, ZEB2

miR-215 ACVR2A, BMPR2, ACVR2B, ZEB2

miR-194 ID4, SMURF1, ZFYVE9, ACVR1, GDF6,

MAPK1, THBS1, PPP2R2C, ACVR2B

BMPR1B, BMPR2, ZEB2

Bladder Cancer miR-192 RB1, DAPK2

miR-215 RB1

miR-194 E2F3, FGFR3, MAPK1, THBS1

MAPK signaling pathway miR-192 MAP3K1, MEF2C, STK4, MAP2K4, FGF7

miR-215 FGFR1, MAP3K7, RAP1B

miR-194 STMN1, ATF2, MEF2C, DUSP9, FGFR3,

MAPK1, RAC1, PAK2, RAP1B, AKT2,

DUSP10, PRKCB1, PDGFA, MAP4K4,

MAP3K3, CRK, PDGFA

P53 signaling pathway miR-192 IGF1, ZMAT3, CYCS, MDM2

miR-215 IGF1, MDM2

miR-194 THBS1, MDM2

Non-small cell lung cancer miR-192 STK4, RB1

miR-215 RB1

miR-194 E2F3, MAPK1, AKT2, PRKCB1

Cell communication miR-192 COL5A1, DSG3, KRT38, GJA7

miR-215 COL5A1

miR-194 THBS1, GJB1, GJB7, COL4A4

Prostate Cancer miR-192 RB1, CREB5, TCF7

miR-215 RB1, FGFR1, CREB5, IGF1, TCF7

miR-194 CDKN1B, E2F3, IGF1R, FOXO1, MAPK1,

AKT2, PDGFA

Cell Cycle miR-192 RB1

miR-215 RB1, MCM3

miR-194 CDKN1B, E2F3, CDC14A

Cell adhesion molecules

(CAMs)

miR-192 ALCAM, MPZ

miR-215 ALCAM, CNTNAP1

miR-194 NRCAM, CDH2, SDC4, CNTNAP2,

ITGA9, CADM1


123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960



ownloaded from


For Peer Review

Colorectal cancer miR-192 FZD9, TCF7, CYCS

miR-215 TCF7

miR-194 FZD5, IGF1R, MAPK1, RAC1, AKT2,

FZD6

Small cell lung cancer miR-192 RB1, CYCS, BIRC4

miR-215 RB1

miR-194 CDKN1B, E2F3, AKT2, COL4A4

Wnt signaling pathway miR-192 FZD9, CTNNBIP1, NFAT5, TCF7

miR-215 MAP3K7,CTNNBIP1, NFAT5, TCF7

miR-194 FZD5, CAMK2G, DAAM1, PPP2R2C,

RAC1, CHD8, PRKCB1, NFAT5, FZD6,

FZD4, PRICKLE2

Pancreatic cancer miR-192 RB1

miR-215 RB1

miR-194 E2F3, MAPK1, RAC1, AKT2

mTOR signaling pathway miR-192 RICTOR, IGF1

miR-215 RICTOR, IGF1

miR-194 HIF1A, RPS6, RHEB, MAPK1, AKT2

Folate Biosynthesis miR-192 DDX50, TYMS

miR-215 DDX50, TYMS

miR-194 TYMS

Focal adhesion miR-192 COL5A1, CAV1, IGF1, BIRC4

miR-215 DIAPH1, COL5A1, RAP1B, MYLK, IGF1

miR-194 TLN2, IGF1R, ITGA9, MAPK1, THBS1,

RAC1, PAK2, RAP1B, AKT2, ARHGAP5,

PRKCB1, ITGB6, PDGFA, CRK, COL4A4

Tight Junction miR-194 RAB13, RAB3B, VAPA, PPP2R2C, CASK,

AKT2, TJAP1, PRKCB1, MYH10

Adherens junction miR-192 TCF7

miR-215 FGFR1, MAP3K7, IQGAP1, TCF7

miR-194 IGF1R, MAPK1, RAC1, IQGAP1

DNA Replication miR-192 TYMS

miR-215 MCM3, TYMS

miR-194 TYMS

Cytokine-cytokine receptor

interaction

miR-192 LEPR, ACVR2A, ACVR2B, BMPR2

miR-215 ACVR2A, BMPR2, ACVR2B

miR-194 IL6ST, TNFSF11, ACVR1, IL10, ACVR2B,

LIF, BMPR2

Apoptosis miR-192 CYCS, BIRC4

miR-194 AKT2, PRKAR1A


123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960



ownloaded from


For Peer Review

Supplementary Table (4): miR-192, miR-194 and miR-215 are predicted to target the

3'UTR of TYMS, MDM2 and ZEB2 based on sequence analysis using the PITA

Algorithm.

Gene miRNA Position Seed

TYMS hsa-miR-192 215 8:1:0

TYMS hsa-miR-215 215 8:1:0

TYMS hsa-miR-194 261 8:1:1

MDM2 hsa-miR-192 370 8:1:1

MDM2 hsa-miR-215 370 8:1:1

MDM2 hsa-miR-194 511 8:1:0

ZEB2 hsa-miR-192 989 8:1:0

ZEB2 hsa-miR-215 989 8:1:0

ZEB2 hsa-miR-194 78 8:1:1


123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960



ownloaded from


Documents

miR-192, miR-194 and miR-215: a convergent microRNA network suppressing tumor progression in renal cell carcinoma