miR-143调控DNMT3B表达对肝细胞癌耐阿霉素敏感性的影响

目的:构建耐药细胞株SMMC-7721/ADM，探讨miR-143在调控肝细胞癌耐阿霉素中的作用机制。方法:采用逐步递增阿霉素浓度法构建耐阿霉素细胞株SMMC-7721/ADM，评估其IC₅₀值；采用荧光定量PCR技术检测miR-143、DNA甲基转移酶3B （DNMT3B）、多耐药基因1（MDR1）变化、Bax和Bcl-2 mRNA表达水平；Western blotting检测DNMT3B蛋白表达；通过转染miR-143 mimics上调耐药细胞株SMMC-7721/ADM中miR-143表达水平后，检测耐药细胞IC₅₀、侵袭能力、细胞凋亡、DNMT3B、MDR1变化。应用脂质体-2000瞬时转染miR-143 mimics于耐药细胞株，观察上述指标变化。结果:成功构建耐药细胞株SMMC-7721/ADM，IC₅₀值升高78.97倍（P<0.01），miR-143表达量降低，DNMT3B、MDR1、Bax、Bcl-2表达量升高（P<0.05~P<0.01）。转染miR-143 mimics后耐药细胞株IC₅₀值降低57%，侵袭能力下降，细胞凋亡增加，DNMT3B表达减少，MDR1 mRNA表达减少（P<0.01）。结论:miR-143可能通过降低DNMT3B表达提高肝细胞癌对阿霉素的敏感性。

Effect of the DNMT3B expression on regulated by miR-143 the sensitivity of the hepatocellular carcinoma resistance to adriamycin

Objective:To establish hepatocellular carcinoma cell line SMMC-7721/ADM resistance to adriamycin,and explore the mechanism of microRNA(miR)-143 in regulating the cell line resistance to adriamycin.Methods:The cell line SMMC-7721/ADM resistance to adriamycin was constructed using the gradual increasing the concentration of adriamycin,and the IC50 value of which was evaluated.The mRNA expression levels of miR-143,DNA methyltransferase 3B(DNMT3B) and multidrug resistance gene 1 (MDR1),Bax and Bcl-2 genes were evaluated using real-time fluorescent quantitative PCR,and the protein level of DNMT3B was determined by Western blotting.After the miR-143 mimics upregulating the miR-143 expression in SMMC-7721/ADM cell line,the IC50 value,invasion ability,cell apoptosis,and levels of DNMT3B,MDR1 were detected.The miR-143 mimics was transiently transfected into the drug resistance cell line using liposome-2000,and the indexes changes were observed.Results:The cell line SMMC-7721/ADM resistance to adriamycin was successfully established,the IC50 value raised 78.97 times (P ＜ 0.01).The expression level of miR-143 decreased,and the expression levels of DNMT3B,MDR1,Bax and Bcl-2 increased,and the differences of which were statistically significant(P ＜0.05 to P ＜ 0.01).After the miR-143 mimics transfecting into the drug resistance cell line,the IC50 value reduced 57％,the invasion ability declined,the cell apoptosis increased,the levels of DNMT3B and MDR1 mRNA decreased,and the differences of which were statistically significant (P ＜ 0.01).Conclusions:The increasing sensitivity of hepatocellular carcinoma to adriamycin may be caused by the miR-143 reducing DNMT3B expression.