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目的:探讨微小RNA(miR)-765对Ⅱ型多磷酸肌醇4-磷酸酶(inositol polyphosphate 4-phosphatase typeⅡ,INPP4B)调控及其对肝细胞癌(hepatocellular carcinoma,HCC)细胞增殖的影响。方法:采用实时定量PCR检测8例HCC组织和癌旁组织以及8组HCC细胞株中的miR-765的表达水平;MTT法检测过表达miR-765对HCC细胞增殖的影响;利用分子生物学技术构建INPP4B的3′-UTR报告基因,分析miR-765对INPP4B的调控作用,然后采用Western blotting法分别检测过表达和沉默miR-765对INPP4B蛋白表达的影响;应用Western blotting法和克隆形成实验探讨特异性抑制INPP4B对HCC细胞株增殖的影响。结果:在HCC癌组织以及HCC细胞株中高表达miR-765(P<0.05)。转染miR-765促进HCC细胞的增殖[(3.78±1.25)vs(2.06±0.47),P<0.05]。miR-765能够显著下调INPP4B的3′-UTR报告基因的荧光表达[(0.42±0.01)vs(1.01±0.01),P<0.05],显著上调INPP4B蛋白的表达[(0.92±0.04)vs(0.42±0.02)、(0.62±0.03),P<0.05]。特异性抑制INPP4B后明显促进HCC细胞的增殖[(238.0±1.73)vs(66.33±5.04),P<0.05]。结论:miR-765通过调控INPP4B的表达来促进HCC细胞的增殖。
Objective: To investigate the effect of microRNA (miR) -765 on the expression of inositol polyphosphate 4-phosphatase type Ⅱ (INPP4B) and its effect on the proliferation of hepatocellular carcinoma (HCC) cells. Methods: Real-time quantitative PCR was used to detect the expression of miR-765 in 8 HCC tissues and adjacent normal tissues and 8 HCC cell lines. The effect of miR-765 overexpression on the proliferation of HCC cells was detected by MTT assay. The 3’-UTR reporter gene of INPP4B was constructed and the regulatory effect of miR-765 on INPP4B was analyzed. The effect of overexpression and silencing miR-765 on INPP4B protein expression was detected by Western blotting. Western blotting and clonogenic assay Specific inhibition of INPP4B on HCC cell proliferation. Results: miR-765 was highly expressed in HCC tissues and HCC cell lines (P <0.05). Transfection of miR-765 promoted the proliferation of HCC cells [(3.78 ± 1.25) vs (2.06 ± 0.47), P <0.05]. miR-765 significantly downregulated the INPP4B expression of 3’-UTR reporter gene (0.42 ± 0.01 vs 1.01 ± 0.01, P <0.05), significantly upregulated the expression of INPP4B protein (0.92 ± 0.04 vs 0.42 ± 0.02), (0.62 ± 0.03), P <0.05]. Specific inhibition of INPP4B significantly enhanced the proliferation of HCC cells [(238.0 ± 1.73 vs 66.33 ± 5.04, P <0.05]. Conclusion: miR-765 promotes the proliferation of HCC cells by regulating the expression of INPP4B.