| | | | | Investigation of transcriptional gene silencing and mechanism induced by shRNAs targeted to RUNX3 in vitro | | | 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 proteinexpression 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.
【基金】：Grants from the National Natural Science Foundation of China, No. 30470967;the Natural Sciences Foundation of Hunan, No. 03Jjy3029
【分类号】：R735.2
【DOI】：CNKI:SUN:ZXXY.0.2008-19-010
【正文快照】：
INTRODUCTION The human runt-related transcription factor 3 gene (RUNX3), a novel tumor suppressor, was originally cloned as AML2 in human leukocythemia by Levanon in 1994 and is located on human chromosome 1p36.1[1]. RUNX3 contains two promoters, p1 and p… | | |
| | |  推荐 CAJ下载  PDF下载 | | | CAJViewer7.0阅读器支持所有CNKI文件格式，AdobeReader仅支持PDF格式 | | | | 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 Cance Research Institute;Nanhua University of South China;Hengyang 421001;Hunan Province;China Department of Oncology;First Affiliated Hospital of Nanhua University of South China;Hengyang 421001;Hunan Province;China | | | 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 proteinexpression 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.
【Keyword】：RNA interference;Short hairpin RNAs;Promoter;DNA methylation;RUNX3;Stomach carcinoma |
| | | | | | 1 | Levanon D, Negreanu V, Bernstein Y, Bar-Am I, Avivi L, Groner Y; AML1, AML2, and AML3, the human members of the runt domain gene-family: cDNA structure, expression, and chromosomal localization. [M];Genomics; 1994年 | | 2 | So K, Tamura G, Honda T, Homma N, Endoh M, Togawa N, Nishizuka S, Motoyama T; Quantitative assessment of RUNX3 methylation in neoplastic and non-neoplastic gastric epithelia using a DNA microarray [M];Pathol Int; 2006年 | | 3 | Ting AH, Schuebel KE, Herman JG, Baylin SB; Short double- stranded RNA induces transcriptional gene silencing in human cancer cells in the absence of DNA methylation [M];Nat Genet; 2005年 | | 4 | Zeng C, He XS, Luo Q, Zhao S, Deng M, Li YN; The expression and the mechanism of the down regulation of RUNX3 in the gastric carcinoma. [M];Shijie Huaren Xiaohua Zazhi; 2006年 |
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