RNA干扰靶向DNMT1基因对乳腺癌MCF-7细胞和RASFF1A基因的影响

Effects of RNA interference targeting DNMT1 gene to breast cancer MCF-7 cell and RASSF1A gene

目的:RASSF1A基因在乳腺癌MCF-7细胞的表达及其对乳腺癌MCF-7细胞周期和凋亡的影响.方法:DNA重组技术合成针对人乳腺癌MCF-7细胞DNA甲基转移酶1(DNMT1)基因3条siRNA(siRNA1,siRNA2,siRNA3),以脂质体2000做为载体将其转入人乳腺癌MCF-7细胞,采用RT-PCR法检测DNMT1mRNA的表达,选择特异性较高的siRNA并以此为标准建立10,30,50nmol/L,3个浓度梯度,采用WesternBlot检测RASSF1A基因的蛋白表达,选出最低且有效浓度已达到基因抑制的最适浓度.采用流式细胞仪检测siRNA抑制DNMT1前后细胞周期和凋亡率的变化.结果:siRNA成功转染了人乳腺癌MCF-7细胞,转染率达到80%;3对siRNA中siRNA2特异性较高;WesternBlot检测终浓度为30nmol/L的siRNA抑制效率较高;细胞周期大部分停滞在G0/G1期,抑制细胞增殖分裂,细胞凋亡率增加.结论:通过RNA干扰可以抑制DNMTI的表达,从而逆转由于启动子高甲基化沉默的抑癌基因RASSF1A的表达,最终抑制乳腺癌细胞的生长.为肿瘤基因治疗提供依据.

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-methylationof anti-oncogene RASSF1A is early event of breast cancer.It inhibits the expression 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（siRNA）for DNMT1 gene were designed and transfected 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 Western Blot and the effective concentration siRNA was selected.The cell cycle and apoptosis rate were analyzed by flow cytometry（FCM）.RESULTS：siRNAtransfected 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 siRNAwas 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.