title: Investigation of transcriptional gene silencing and mechanism induced by shRNAs targeted to RUNX3 in vitro.
abstract: OBJECTIVE
  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 G(0)/G(1) 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Cancer Research Institute, Nanhua University of South China, Hengyang 421001, Hunan Province, China.
authors.forename: Xue-Zhi
authors.initials: XZ
authors.lastname: Feng
authors.forename: Xiu-Sheng
authors.initials: XS
authors.lastname: He
authors.forename: Ying-Zhi
authors.initials: YZ
authors.lastname: Zhuang
authors.forename: Qiao
authors.initials: Q
authors.lastname: Luo
authors.forename: Jun-Hao
authors.initials: JH
authors.lastname: Jiang
authors.forename: Shuai
authors.initials: S
authors.lastname: Yang
authors.forename: Xue-Fang
authors.initials: XF
authors.lastname: Tang
authors.forename: Ju-Lin
authors.initials: JL
authors.lastname: Liu
authors.forename: Tao
authors.initials: T
authors.lastname: Chen
mesh_terms: D001374:Azacitidine / Q000031:analogs & derivatives / Q000494:pharmacology; D015153:Blotting, Western; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D050677:Core Binding Factor Alpha 3 Subunit / Q000235:genetics* / Q000378:metabolism; D019175:DNA Methylation; D015254:DNA Modification Methylases / Q000037:antagonists & inhibitors; D000077209:Decitabine; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D007150:Immunohistochemistry; D011401:Promoter Regions, Genetic; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013274:Stomach Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D013997:Time Factors; D014158:Transcription, Genetic / Q000187:drug effects; D014162:Transfection
chemical_list: D050677:Core Binding Factor Alpha 3 Subunit; D004791:Enzyme Inhibitors; D012333:RNA, Messenger; D034741:RNA, Small Interfering; C493729:Runx3 protein, human; D000077209:Decitabine; D015254:DNA Modification Methylases; D001374:Azacitidine