miR-101-3p靶向调控SOX9影响肝癌细胞增殖凋亡的研究

Study on the effect of miR-101-3p targeting SOX9 on proliferation and apoptosis of hepatocellular carcinoma cells

目的探讨miR-101-3p对肝癌细胞增殖凋亡的影响及其对靶基因性别决定区Y框蛋白9(sex determining region Y-box 9,SOX9)的调控作用。方法肝癌细胞转染miR-101-3p模拟物(miR-101-3p mimics组)、模拟物阴性对照(mimics control组)、SOX9小干扰RNA(SOX9 siRNA组)、siRNA对照(siRNA control组),同时以只加入转染试剂的细胞为对照组,RT-PCR检测细胞中miR-101-3p水平,噻唑蓝(MTT)检测细胞增殖情况,流式细胞术检测细胞凋亡情况,Western blotting检测细胞中活化的含半胱氨酸的天冬氨酸蛋白水解酶3(Cleaved Caspase-3)、SOX9表达,靶基因预测库预测miR-101-3p靶基因,荧光素酶报告基因鉴定靶基因。结果miR-101-3p mimics能够促进肝癌细胞中miR-101-3p表达,SOX9 siRNA能够抑制肝癌细胞中SOX9的表达。miR-101-3p mimics组细胞凋亡率高达(35.69±5.27)%,细胞存活率只有(76.34±4.63)%,细胞中Cleaved Caspase-3表达增多。靶基因预测库和双荧光素酶报告基因预测并鉴定miR-101-3p的靶基因是SOX9。miR-101-3p mimics能够抑制SOX9 mRNA和蛋白表达。SOX9 siRNA组细胞中SOX9蛋白表达下降,Cleaved Caspase-3蛋白水平升高,细胞凋亡率高达(29.58±2.58)%,而细胞存活率只有(69.47±5.26)%。结论miR-101-3p能够负调控靶基因SOX9,从而促进肝癌细胞凋亡,抑制肝癌细胞增殖。

Objective To investigate the effect of miR-101-3 p on proliferation and apoptosis of hepatocellular carcinoma cells and the regulation of sex determining region Y-box 9（ SOX9）. Methods Hepatocellular carcinoma cells were transfected with miR-101-3 p mimics（ miR-101-3 p mimics group）,mimics negative control（ mimics control group）,SOX9 small interfering RNA（ SOX9 siRNA group） and siRNA control（ siRNA control group）. At the same time,the cells transfected with transfection reagent were used as control group,RT-PCR was used to detect the level of miR-101-3 p in cells. MTT was used to detect the proliferation of cells,and apoptosis was detected by flow cytometry,expressions of Cleaved Caspase-3 and SOX9 in cells were detected by Western blotting. miR-101-3 p target gene was predicted by target gene prediction library,luciferase reporter gene targeting gene. Results miR-101-3 p mimics could promote the expression of miR-101-3 p in hepatocellular carcinoma cells,SOX9 siRNA could inhibit the expression of SOX9 in hepatocellular carcinoma cells. The apoptosis rate of miR-101-3 p group mimics was（ 35. 69 ± 5. 27） %,the cell survival rate was only（ 76.34±4.63） %,the expression of Cleaved Caspase-3 in cells was increased. Target gene prediction library and double luciferase reporter gene predicted and identified the target gene of miR-101-3 p was SOX9. miR-101-3 p mimics could inhibit the expression of SOX9 mRNA and protein. The expression of SOX9 protein was decreased in SOX9 siRNA group,the level of Cleaved Caspase-3 protein was elevated. The apoptosis rate was（ 29.58 ± 2.58） %,the cell survival rate was（ 69. 47 ± 5. 26） %. Conclusion miR-101-3 p can negatively regulate the target gene SOX9 and promote the apoptosis of hepatocellular carcinoma cells,inhibite proliferation of hepatocellular carcinoma cells.