20071116_mtDNA workshop

Idenfitied Differentially Expressed Genes in

Keratoconus

Ji Eun Lee, Jong Soo Lee

Department of Ophthlamology, Graduate School of Medicine, Pusan National University

20071116_mtDNA workshop

Authors have no financial interest in any materials discussed in this article.

Purpose

• It is valuable to examine the genes of the keratocytes that are involved

in the thinning of the cornea because investigation of the fundamental

pathogenesis is important to understand the disease of the keratoconus.

• The purpose of this study is to evaluate the pathogenesis of keratoconus

through differentially expressed genes in human keratocyte of

keratoconus.

Methods

• Culture of human keratocyte

• mRNA isolation

• cDNA synthesis

• Annealing Control Primer (ACP)-

based GeneFisingTM PCR

• Cloning and Sequencing

• RT-PCR

• Quantitative Real-Time PCR

Figure 1. ACPTM structure. APCTM technology provides a primer with annealing specificity

to the temple and allows only real products to be amplifies, such that it enables the

researchers to find only real products as a result. A: core sequence - annealing at the first

stage of PCR (targeting). B: universal sequence - annealing at the second stage of PCR. C:

regulator- regulating the functions of A and B.

Results

500bp

1000bp1 : 정상각막

2 : 원추각막

A EBA A

1 2

E

1000bp

500bp

1 2

A

1 2

C

1 2

D

1 2

B

1 2 1 2 1 2

F G H

Figure 2. ACP-based PCR product of the eight verified differentially expressed genes

showing increased or decreased levels of expression in keratoconus (arrows). 1: normal

cornea. 2: keratoconus. A: BMP4. B: ACTA 2. C: CFL 1, D: GRCC 10, E: TIMP 3, F:

MRVI1, G: TIMP1, H: SSTR1. 

Results

GAPDH

BMP4

1 2

GAPDH

ACTA2

1 2

GAPDH

CFL1

1 2

GAPDH

GRCC10

1 2

GAPDH

TIMP3

1 2

GAPDH

MRVI1

1 2

GAPDH

TIMP1

1 2

GAPDH

SSTR1

1 2

Figure 3. Depicted are RT-PCR products of the eight verified genes and a housekeeping gene

(GAPDH). 1: normal cornea. 2: keratoconus.

Cytoskeleton - CFL1, GRCC10, SSTR1, TIMP1, TIMP3

Wound healing - ACTA2

Apoptosis - BMP4, CFL1, MRVI1

Results

Figure 4. Quantitative real-time polymerase chain reaction. Quantitative real-time polymerase chain reaction of the eight verified genes using GAPDH as endogenous control showed that BMP4 (A), CFL1 (B), MRVI1 (C) were increased by 1.6, 3.3, and 11 fold, respectively, whereas ACTA2 (D), GRCC10 (E), TIMP3 (F), TIMP1 (G), and SSTR1 (H) were decreased by 4.5, 2.7, 14, 8.5, and 1.8 fold, respectively in keratoconus relatively to norma l cornea.

Conclusions

• We found differential expression of genes related to apoptosis, as well as those

related to cytoskeleton structure and wound healing in keratoconus, using

GeneFishingTM PCR.

• This was based on the PCR method using the mRNA of keratocytes in normal

cornea and keratoconus.

• We also confirmed the importance of the apoptosis, cytoskeleton, and wound

healing of keratocytes as an important cause of keratoconus through the

differential expressions of genes.

• We anticipate that gene therapy techniques using such genes will suppress the

progress and side effects of keratoconus in the future.