ZBTB20 is involved in liver regeneration after partial hepatectomy in mouse

Hepatobiliary & Pancreatic Diseases International

48 • Hepatobiliary Pancreat Dis Int，Vol 13，No 1 • February 15，2014 • www.hbpdint.com

Original Article / Liver

Author Affiliations: Department of General Surgery, Xinhua Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200092, China (Weng MZ, Zhuang PY, Hei ZY, Lin PY, Chen ZS, Liu YB, Quan ZW and Tang ZH)

Corresponding Author: Zhao-Hui Tang, MD, PhD, Department of General Surgery, Xinhua Hospital, Shanghai Jiaotong University School of Medicine, No. 1665 Kongjiang Street, Shanghai 200092, China (Tel/Fax: 86-21-25078999; Email: [email protected])

© 2014, Hepatobiliary Pancreat Dis Int. All rights reserved.doi: 10.1016/S1499-3872(14)60006-0

BACKGROUND: A better understanding of the molecular mechanisms in liver regeneration holds promise for exploring the new potential therapy for liver failure. The present study was to investigate the role of zinc finger and BTB domain-containing protein 20 (ZBTB20), a potential factor associated with liver regeneration, in a model of 70% hepatectomy in mice.

METHODS: Parameters for liver proliferation such as liver/body ratio and BrdU positivity were obtained via direct measurement and immunohistochemistry. The levels of zinc fingers and homeoboxes 2 (ZHX2), ZBTB20, alpha-fetoprotein (AFP) and glypican 3 (GPC3) transcripts in the regenerating liver tissue of a 70% hepatectomy rodent model were monitored by real-time PCR analysis at different time points. Knockdown of ZBTB20 was performed to characterize its regulatory function.

RESULTS: A negatively regulating relationship between ZHX2, ZBTB20 and AFP, GPC3 was revealed from 24 to 72 hours after 70% hepatectomy. ZBTB20 appears to negatively regulate AFP and GPC3 transcription since the knockdown of ZBTB20 promoted the proliferation of hepatocytes and the expression of AFP and GPC3.

CONCLUSION: In addition to AFP, GPC3 and ZHX2, ZBTB20 is a new regulator in liver regeneration and the decrease of ZBTB20 expression following 70% hepatectomy promotes AFP and GPC3 expression.

(Hepatobiliary Pancreat Dis Int 2014;13:48-54)

KEY WORDS: partial hepatectomy; liver regeneration; ZBTB20; mouse

Introduction

The liver can functionally recover from hepatic injury such as surgery, trauma, poisoning, infection, and so on, which indicates that the liver

possesses remarkable regenerative capabilities. A better understanding of the molecular mechanisms in liver regeneration helps to find a potential effective therapy for liver failure. Partial hepatectomy of rodent model has been extensively utilized for investigating this highly precise molecular regulation, which has led to the identification of many factors necessary for liver regeneration.[1]

Alpha-fetoprotein (AFP), a major plasma protein produced by the yolk sac and the liver during fetal development, is thought to be the fetal form of serum albumin.[2, 3] Glypicans are a small family of glycosylphosphatidylinositol-anchored heparin sulfate proteoglycans,[4] which is associated with cell growth, development and responses to various growth factors. Among different glypicans, X-linked glypican 3 (GPC3) has been associated with certain tumors such as breast and ovarian tumors and mesotheliomas.[5-7] AFP and GPC3 are expressed in fetal liver but not in adult liver, they are reactivated in hepatocellular carcinoma and therefore, are the markers of hepatocellular carcinoma.[8] Furthermore, their levels are elevated in the regenerating liver and they are the markers of liver regeneration.[9-11]

The correlation between zinc fingers and homeoboxes 2 (ZHX2) and AFP was noticed in BALB/cJ mice.[12, 13] Loss of the ZHX2 gene in BALB/cJ mice leads to incomplete silencing of its target AFP, and the AFP mRNA level is about 10-20 times more in BALB/cJ mice than that in other strains. This suggests that ZHX2 negatively regulates AFP. In addition, Morford et al[14] reported that GPC3 was up-regulated in the liver of BALB/cJ mice and this trend could be reversed by transfecting the ZHX2 gene into BALB/cJ mice, which indicates that ZHX2 could also repress GPC3 expression.

ZBTB20 is involved in liver regeneration after partial hepatectomy in mouseMing-Zhe Weng, Peng-Yuan Zhuang, Zhen-Yu Hei, Pei-Yi Lin, Zhi-Sheng Chen,

Ying-Bin Liu, Zhi-Wei Quan and Zhao-Hui Tang

Shanghai, China

ZBTB20 in liver regeneration

Hepatobiliary Pancreat Dis Int，Vol 13，No 1 • February 15，2014 • www.hbpdint.com • 49

Another zinc finger family protein−zinc finger and BTB domain-containing protein 20 (ZBTB20) was also found to be activated in fetal liver and its expression inversely correlated with AFP gene expression.[15] However, it is not clear whether ZBTB20 plays a role in the regulation of AFP and GPC3 gene transcription during liver regeneration.

The present study was to elucidate some of the molecular mechanisms of liver regeneration. We used a partial hepatectomy in mice to observe the relationships between regeneration-related genes (AFP and GPC3) and two zinc finger protein genes (ZHX2 and ZBTB20).

MethodsZBTB20 siRNA-expressing adenovirus

Adenoviruses were created with the Ad Easy System from Stratagene Biotechnology (La Jolla, CA, USA). siRNA-expressing adenovirus targeting mouse ZBTB20 was constructed according to the manufacturer's instructions. Briefly, one pair of oligonucleotides targeting mouse ZBTB20 mRNA was synthesized by Sangon Biotech (Shanghai, China). The dsDNA was ligated between the BamHI and HindIII sites on the pShuttle containing H1 promoter and GFP sequences. The control (mock) vector was constructed by inserting oligonucleotides that include a siRNA with limited homology to sequences in the mouse genome. Adenoviral DNA was prepared on a large scale in Escherichia coli DH5α, generating Ad/ ZBTB20 siRNA and Ad/siRNA. All adenoviruses were packaged in HEK293 cells and purified using Adeno-X ™ purification kit (BD Biosciences Clontech, Mountain View, CA, USA). Viral titers were determined using Adeno-X™ rapid titer kit from the same company.

Cell culture and transfection

Mouse hepatocyte cell line TIB-73 (from the Chinese Academy of Sciences, Shanghai, China) was used. The cells were cultured in RPMI 1640 medium (HyClone Laboratories Inc., Logan, UT, USA) supplemented with 10% fetal bovine serum (HyClone Laboratories Inc., Logan, UT, USA), 100 U/mL penicillin and 100 mg/ mL streptomycin, and were incubated in a 5% CO2

atmosphere at 37 ℃. TIB-73 cells were infected with the Ad/ZBTB20 siRNA at 50 PFU/cell, and TIB-73 cells infected with Ad/siRNA were used as controls. Adenovirus generation was confirmed by the expression of GFP. For cell growth curves, TIB-73, TIB-73+Ad/siRNA and TIB-73+Ad/ZBTB20 siRNA were harvested and reseeded at 1×104 cells/well in 12-well plates. The total cell number was determined every day with a

Fig. 1. Mouse liver. A: The anatomy of mouse liver. LLL, LML and RML were resected. B: Liver morphology after major hepatectomy. LLL: left lateral lobe; LML: left middle lobe; RML: right middle lobe; RUL: right upper lobe; RLL: right lower lobe; CL: caudate lobe; GB: gallbladder; PCL: posterior caudate lobe; ACL: anterior caudate lobe.

hematocytometer and by using an inverted microscope (Olympus, Tokyo, Japan).

Animal husbandry and surgery

C57BL/6J mice (age 6-10 weeks, body weight 20-30 g) were obtained from the Shanghai Laboratory Animal Center of the Chinese Academy of Sciences (Shanghai, China). The mice were housed in groups of three to five in a vivarium maintained on a 12-hour light/dark cycle with a temperature of 22±1 ℃ and a relative humidity of 50±5%. All mice were divided into two groups: major hepatectomy (70% partial hepatectomy) group and minor hepatectomy (5% partial hepatectomy) group (n=35 in each group). Major hepatectomy was performed under general anesthesia (4% chloral hydrate at a dose of 8 µL/g, intraperitoneal injection) and the left lateral, left middle and right middle lobes were removed (Fig. 1). Minor hepatectomy was performed with the resection of the left middle lobe was resected. The removed liver during the surgery was considered as sample at 0 hour. At each time point (2, 4, 24, 48, 72, 96 and 168 hours) after surgery, 5 animals were sacrificed by CO2 inhalation and liver tissues were harvested. All procedures were carried out in accordance with the guidelines of the Ethics Committee of Xinhua Hospital, Shanghai Jiaotong University School of Medicine.

Liver tissue collection, histology and immunohisto-chemistry

At each time point after the surgery, regenerating liver samples were collected and liver weights were measured. The liver/body weight ratio (%) was calculated to evaluate regeneration. Bromodeoxyuridine (BrdU) labeling was used to identify hepatocytes within S-phase. Briefly, 100 mg BrdU (Dako Corp., Carpinteria, CA, USA) per kg of body weight was injected intraperitoneally 30 minutes before the animals were euthanized. The parafin-embedded liver tissue was



cut into 6-µm sections, deparafinized, and cooked in a microwave oven for 10 minutes in Tris-buffered citric acid (pH=6) for antigen retrieval. BrdU incorporation was detected immunohistologically using a monoclonal anti-BrdU antibody (Dako Corp., Carpinteria, CA, USA) according to the supplied protocol. Hepatocyte proliferation was measured by calculating the mean number of hepatocytes with positive BrdU nuclear staining in eight 100× microscopic fields.

Quantitative real-time PCR

Total RNA was extracted from cells using Trizol reagent kit (Gibco BRL, Gaithersburg, MD, USA) according to the manufacturer's instructions. After quantification, complementary DNA (cDNA) was synthesized from 2 µg of total RNA using a Takara RNA PCR kit (Takara Bio Inc., Dalian, China) according to manufacturer's instructions. The primers were designed by Primer Premier Version 5.0 and synthesized by Sangon Biotech (Shanghai, China) (Table). The PCR was performed as follows: initial denaturation at 95 ℃ for 5 minutes, 40 cycles of 20 seconds at 94 ℃, 20 seconds at 61 ℃ for primer annealing extension. β-actin was used as control (mock).

Western blotting analysis

Total protein was extracted from the treated cells using RIPA lysis buffer (Beyotime Biotechnology, Haimen, China) supplemented with 1 mmol/L phenyl-methanesulfonyl fluoride. The protein concentration was measured with BCA protein assay system (Beyotime Biotechnology, Haimen, China). Total proteins (40-50 µg) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride (PVDF) membranes. The blots were blocked with 5% skim milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) for

Table. Primer sequences used for the real-time PCR analysis

Primer Sense (5'-3') Anti-sense(5'-3')

AFP TCT GCT GGC ACG CAA GAA G

TCG GCA GGT TCT GGA AAC TG

GPC3 CGC TTT GCC GGG CTA CAT CTG C

CCG TTC CTT GCC GCC TTC TGG

ZHX2 TGG AAG CGA GGC GGC ACA TCA G

CCG GCT CCA GCT ACC CCA CTT CTC

ZBTB20 AGC AAC ACA CAG GTC ATT GGC

CGG GTT GGG TAG TGA AGA GG

β-actin CGT TGA CAT CCG TAA AGA CC

AAC AGT CCG CCT AGA AGC AC

AFP: alpha-fetoprotein; GPC3: glypican 3; ZHX2: zinc fingers and homeo-boxes 2; ZBTB20: zinc finger and BTB domain-containing protein 20.

2 hours at room temperature, incubated with primary antibodies against ZBTB20 (Abcam Inc., Cambridge, UK) (1:500), AFP (Abcam Inc., Cambridge, UK) (1:1000), and GPC3 (Abcam Inc., Cambridge, UK) (1:1000). β-actin (Zhongshan Golden Bridge Biotech, Beijing, China) (1:10 000) was used as a loading control. The membranes were incubated with the respective primary antibodies overnight at 4 ℃. They were washed in TBST for three times and then incubated with horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG (Zhongshan Golden Bridge Biotech, Beijing, China) (1:10 000) for 1 hour at room temperature. The protein bands were detected using the Super Signal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific Inc., Rockford, IL, USA) and visualized using a VersaDoc Imaging System (Bio-Rad Laboratories Co., Ltd., Hercules, CA, USA). Densitometric analysis was performed using Quantity One Software v4.62 (Bio-Rad Laboratories Co., Ltd., Hercules, CA, USA) and the results were presented as the mean of three independent experiments.

Statistical analysis

Data were expressed as mean±SD. SPSS Software (SPSS, Chicago, IL, USA) was used for statistical analyses. Analysis of variance (ANOVA) was used for comparisons. All P values were two-tails and considered to be statistically significant when P<0.05.

ResultsLiver regeneration after partial hepatectomy

The ratio of the liver to body weight was significantly increased at the point of 48 hours post major hepatectomy compared to that at the point of 24 hours. However, the ratio did not change significantly in the minor hepatectomy group. Seven days after surgery, there was no significant difference in the liver/body weight ratio between the two groups, indicating that the recovery of the rodent liver was about one week (Fig. 2). BrdU staining showed that the number of BrdU positive cells was significantly increased in the liver tissue at 24, 72, and 168 hours after hepatectomy compared with that in the controls. BrdU positive cells peaked at 72 hours after hepatectomy (Fig. 3).

AFP and GPC3 mRNA transcriptions were increased in parallel with liver regeneration

AFP mRNA level exhibited a gradual increase in the major hepatectomy group and reached a significant level after 24 hours compared with the minor hepatectomy group. AFP peaked at 72 hours after major hepatectomy



and then dropped rapidly toward normal value (Fig. 4A). The trend of GPC3 mRNA expression was similar to that of AFP. In the minor hepatectomy group, the AFP

Fig. 3. BrdU immunostaining of regenerative liver tissue (original magnification ×100). A: control; B : liver tissue obtained 24 hours after hepatectomy; C: liver tissue obtained 72 hours after hepatectomy; D: liver tissue obtained 168 hours after hepatectomy; E: the number of BrdU positive hepatocytes of liver tissue from control, 24, 72 and 168 hours after hepatectomy (*: P<0.05). Eight fields of each histological section in liver tissue were photographed.

Fig. 2. Liver/body weight ratios of major and minor hepatectomy groups (*: P<0.05; #: P<0.05, compared with 2 hours after surgery).

Fig. 4. mRNA levels as measured by quantitative PCR (*: P<0.05). A: time-course of AFP mRNA expression; B: time-course of GPC3 mRNA expression; C: time-course of ZHX2 mRNA expression; D: time-course of ZBTB20 mRNA expression. All RNA levels were normalized to β-actin.

and GPC3 mRNA levels did not change significantly at the different time points (Fig. 4A, B).



ZHX2 and ZBTB20 mRNA transcriptions

On the contrary, both ZHX2 and ZBTB20 mRNA transcriptions in the major hepatectomy group were decreased after partial hepatectomy and reached a significant level in 24 hours compared with the minor hepatectomy group in which there were no significant changes at each time point. Both ZHX2 and ZBTB20 mRNA transcriptions increased back toward normal in 72 hours after major hepatectomy (Fig. 4C, D).

ZBTB20 knockdown increased AFP and GPC3 expressions and enhanced the proliferation of TIB-73 hepatocytes

To verify that ZBTB20 was a negative regulator of AFP and GPC3, we conducted a knockdown experiment with the siRNA technique. TIB-73 cells were stably transfected with ZBTB20 siRNA-expressing adenovirus, which was confirmed by detection of GFP (Fig. 5A). The marked knockdown of ZBTB20 (>85%) at mRNA (Fig. 5B) and protein (Fig. 5C) levels were detected by real-time PCR and Western blotting analysis.

In the cells with confirmed knockdown of ZBTB20, we observed a significant increase in AFP and GPC3 (Fig. 5D). Knockdown of ZBTB20 also promoted the proliferation of TIB-73 cells. The cell number in the three groups (TIB-73+Ad/ZBTB20 siRNA, TIB-73+Ad/siRNA, and TIB-73) was calculated every day. The growth rate of cells with confirmed knockdown of ZBTB20 was significantly higher than that of the other two groups in 24 hours after transfection (Fig. 6).

Fig. 6. Growth curves of mock, Ad/siRNA and Ad/ZBTB20 siRNA treated TIB-73 cells.

Fig. 5. Effect of Ad/ZBTB20 siRNA on ZBTB20 expression in TIB-73 cells. A: TIB-73 cells were infected with Ad/ZBTB20 siRNA at a multiplicity of infection of 50 in which nearly all the cultured TIB-73 cells were GFP-positive under fluorescent microscope; B: ZBTB20 mRNA was quantified by real-time PCR. All RNA levels were normalized to mock group (β-actin); C: ZBTB20 protein was quantified by Western blotting; D: The AFP and GPC3 levels in TIB-73 cells after infection were quantified by Western blotting.

DiscussionTo our knowledge, this is the first study to investigate the association of ZBTB20 and liver regeneration. We found that ZBTB20 significantly repressed AFP and GPC3 expressions as well as liver regeneration. ZBTB20 knockdown significantly increased AFP and GPC3 expressions and enhanced hepatocytes proliferation.

Liver regeneration is a fundamental response to hepatic damage and has been extensively studied since the two-thirds partial hepatectomy model in rodents was described.[1] The advantage of this animal model over administration with carbon tetrachloride (CCl4) is that in partial hepatectomy, all hepatocytes are exposed to the same stimuli resulting in less heterogeneity. The liver regeneration generally reached completeness within one week in this study, which is consistent with what



was reported in the literature.[1]

Several factors have been identified to be necessary for quiescent mature hepatocytes to rapidly reenter the cell cycle and proliferate to restore the original liver mass. AFP was reported at a high level in the visceral endoderm of the yolk sac and fetal liver, but rapidly repressed after birth and remained at an extremely low level in normal liver tissue.[2, 3] However, this repression is reversible as the AFP gene can be re-activated during liver regeneration and hepatocellular carcinoma. Thus, it has been proposed that AFP transcription level is correlated to the proliferation of hepatocytes.[16]

GPC3 was discovered as a potential serological and histochemical marker for hepatocellular carcinoma.[17] Because the expression of the GPC3 gene is active in fetal liver and hepatocellular carcinoma but silent in normal adult liver, it seems reasonable that it shared the same pattern of regulation with AFP.[18] In accordance with other's findings,[10, 11] we found that AFP and GPC3 mRNA levels began to significantly increase in 24 hours after major hepatectomy and peaked at 72 hours, then dropped dramatically from 72 to 168 hours.

On the contrary, we observed that ZHX2 level was significantly declined at 24 and 48 hours in the major hepatectomy group whereas the levels of AFP and GPC3 elevated significantly. At 48 hours after operation, the expression of ZHX2, similar to that of AFP and GPC3, returned to the normal level. Our findings suggested that liver regeneration induced the repression of ZHX2 and inversely promoted the expression of AFP and GPC3 (24 to 72 hours post-hepatectomy), which was consistent with previous studies and confirmed that ZHX2 is an important repressor of AFP in postnatal mouse liver.[12, 13]

ZBTB20 was initially described as one of a series of genes expressed in human dendritic cells.[19] The proteins belong to the ZBTB family contain an N-terminal BTB (Broad complex, tramtrack, bric-a-brac) domain and multiple C-terminal Kruppel-like C2H2 zinc fingers which often act as transcriptional repressors.[20] Wang et al[21] have reported that ZBTB20 increased in hepatocellular carcinoma and was inversely related to AFP expression in tumor tissue, which was in keeping with our findings.

The changes in mRNA expression observed after partial hepatectomy prompted us to speculate that ZBTB20 was another negative regulator of AFP and GPC3 genes in addition to ZHX2. To verify this, siRNA targeting ZBTB20 mRNA was constructed and introduced into TIB-73 cells by adenovirus. Knockdown of ZBTB20 in TIB-73 cells led to significantly higher expression of AFP and GPC3 and higher cell growth rate (24 hours after transfection).

There was still some deficiency in this work. AFP

activation in C57BL/6J mice is lower than that in other mouse strains due to a genetically unlinked transacting locus, Afr2 (alpha-fetoprotein regulator 2),[22] thus we are going to investigate the other mouse strains. What's more, the mechanism of how hepatectomy starts repression of zinc finger protein genes is still worth exploring.

In summary, there were inverse correlations between ZHX2, ZBTB20 and AFP, GPC3 at 24 to 72 hours after major hepatectomy in our rodent model. We found that in liver regeneration after 70% hepatectomy, the decrease of ZBTB20 expression would consequently promote the expression of AFP and GPC3 as well as hepatocytes proliferation.

Contributors: TZH proposed the study. WMZ and TZH performed research and wrote the first draft. WMZ collected and analyzed the data. All authors contributed to the design and interpretation of the study and to further drafts. TZH is the guarantor.Funding: This study was supported by a grant from the Shanghai Municipal Commission of Education Scientific Research and Innovation Project (11zz107).Ethical approval: This study was approved by Ethics Committee of Xinhua Hospital, Shanghai Jiaotong University School of Medicine.Competing interest: No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.

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Received August 30, 2013Accepted after revision November 17, 2013

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ZBTB20 is involved in liver regeneration after partial hepatectomy in mouse