Effects of RNA interference targeting DNMT1 gene to breast cancer MCF-7 cell and RASSF1A gene
AIM: DNA methyltransferase 1 gene expression is quite high in many tumors cell, but quite a few in adult normal cell, so DNA methyltransferase 1 have close relationship with tumorous emergence. While promoter hyp-methylation of anti-onco-gene RASSF1A is early event of breast cancer. It inhibits the ex pression of DNMT1 by RNA interference for this investigate ; then detect the expression of RASSF1A in breast cancer cell MCF-7 and the effect of cell cycle and apoptosis of breast cancer cell MCF-7. METHODS: Three pairs of small interfering RNA(siR NA) for DNMT1 gene were designed and transferred into breast cancer cell MCF-7. The mRNA expression of DNMT1 gene was examined by real-time quantitative polymerase chain reaction ( RT PCR) and differential siRNA was selected by RT-PCR. Breast cancer cell lines MCF-7 were treated with 20, 30 and 50 nmol/L siRNA. RASSF1A gene protein expression was detected by West ern Blot and the effective concentration siRNA was selected. The cell cycle and apoptosis rate were analyzed by flow cytometry (FCM). RESULTS: siRNA transfected breast cancer cell MCF 7 successfully and transfection efficiency exceeds eighty percent. The number of G0/G1 cell was increased, and 5-Aza-CdR blocked the cell cycle at G1/G0 phase, the apoptotic rate was also increased significantly, ( RT-PCR) detected differential siRNA was siRNA two. Western-blot detected protein of RASSF1A gene in breast cancer cell line recovered expression and the effective concentration siRNA was 30 nmol/L. CONCLUSION: RNA interference can check the expression of DNMT1 , it can reverse that promoter methylation to make RASSF1A gene express again and restrain the growth of breast cancer cell MCF-7.
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万方数据
维普
