Gastric Cancer
Copyright ©2008 The WJG Press and Baishideng. All rights reserved.
World J Gastroenterol. May 21, 2008; 14(19): 3006-3014
Published online May 21, 2008. doi: 10.3748/wjg.14.3006
Investigation of transcriptional gene silencing and mechanism induced by shRNAs targeted to RUNX3 in vitro
Xue-Zhi Feng, Xiu-Sheng He, Ying-Zhi Zhuang, Qiao Luo, Jun-Hao Jiang, Shuai Yang, Xue-Fang Tang, Ju-Lin Liu, Tao Chen
Xue-Zhi Feng, Xiu-Sheng He, Qiao Luo, Jun-Hao Jiang, Shuai Yang, Xue-Fang Tang, Ju-Lin Liu, Tao Chen, Cancer Research Institute, Nanhua University of South China, Hengyang 421001, Hunan Province, China
Xue-Zhi Feng, Ying-Zhi Zhuang, Department of Oncology, First Affiliated Hospital of Nanhua University of South China, Hengyang 421001, Hunan Province, China
Author contributions: Feng XZ designed and performed the research and wrote the paper; He XS contributed the acquisition of funding; He XS and Zhuang YZ guided the research and revised the paper critically for intellectual content; Qiao L contributed technology and materials support; Jiang JH, Yang S, Tang XF, Liu JL and Chen T checked the statistical analysis of data.
Correspondence to: Xiu-Sheng He, MD, Cancer Research Institute, Nanhua University of South China, Hengyang 421001, Hunan Province, China. hexiusheng@hotmail.com
Telephone: +86-734-8281725
Fax: +86-734-8281305
Received: December 29, 2007
Revised: March 16, 2008
Published online: May 21, 2008
Abstract

AIM: To investigate transcriptional gene silencing induced by short hairpin RNAs (shRNAs) that target gene prompter regions of RUNX3 gene, and whether shRNAs homologous to DNA sequences may serve as initiators for methylation.

METHODS: According to the principle of RNAi design, pSilencer3.1-H1-shRNA/RUNX3 expression vector was constructed, The recombinant plasmid shRNA was transfected into human stomach carcinoma cell line SGC7901 with Lipofectamine 2000. Then, the positive cell clones were screened by G418. The mRNA and protein expression level of RUNX3 in the stable transfected cell line SGC7901 were determined by RT-PCR, Western blotting and immunocytochemistry. Characteristics of the cell lines including SGC7901, pSilencer3.1-H1/SGC7901 and pSilencer3.1-H1-shRNA/RUNX3/SGC7901 were analyzed with growth curves, clone formation rate and cell-cycle distribution. The activated level of RUNX3 was examined after treatment with the different density of 5’-aza-2’-deoxycytidine (5-Aza-CdR) by using semi-quantitative RT-PCR and Western blotting.

RESULTS: In the cell line SGC7901 transfected with pSilencer3.1-H1-shRNA/RUNX3, mRNA and protein expression of the RUNX3 gene was lost identified by RT-PCR, Western blotting and immunocytochemistry assay. The growth of pSilencer3.1-H1-shRNA/ RUNX3/SGC7901 cells without expression of RUNX3 was the fastest (P < 0.05), its rate of clone formation was the highest (P < 0.01), and the cell distribution in G0/G1 and S/M phases was lowest and highest, respectively (P < 0.05), compared with that of the transfected pSilencer3.1-H1 and non-transfected cells. Through RT-PCR and Western blot assay, inactivated RUNX3 could not be reactivated by 5-Aza-CdR.

CONCLUSION: We found that, although shRNAs targeted to gene prompter regions of RUNX3 could effectively induce transcriptional repression with chromatic changes characteristic of inaction promoters, this was independent of DNA methylation, and the presence of RNA-dependent transcriptional silencing showed that RNA-directed DNA methylation might be an existing gene regulatory mechanism relative to the methylated in humans.

Keywords: RNA interference, Short hairpin RNAs, Promoter, DNA methylation, RUNX3, Stomach carcinoma