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目的:研究siRNA沉默RhoC基因表达对人肝癌细胞BEL7402凋亡的影响及其机制,为肝癌的基因治疗提供实验依据。方法:构建RhoC-siRNA真核表达载体pU6mRFPRhoC-siRNA,转染BEL7402细胞,激光共聚焦显微镜检测转染效率,RT-PCR和Western blotting鉴定RhoC基因沉默效果;流式细胞术、琼脂糖凝胶电泳和瑞氏染色检测BEL7402细胞凋亡,RT-PCR检测细胞凋亡相关基因Bcl-2和Bax的表达。结果:成功构建pU6mRFPRhoC-siRNA重组载体,转染BEL7402细胞的效率为70%,RT-PCR和Western blotting检测RhoC基因沉默效率分别为85%和82%。pU6mRFPRhoC-siRNA转染组BEL7402细胞凋亡显著高于未转染BEL7402细胞和pU6mRFP Scramble-siRNA转染组BEL7402细胞[(21.00±2.23)%vs(6.47±1.64)%、(4.63±0.47)%,P<0.01)],pU6mRFPRhoC-siRNA转染组BEL7402细胞DNA呈典型的“梯状”条带,瑞氏染色见转染组BEL7402细胞中有大量凋亡细胞。pU6mRFPRhoC-siRNA转染组BEL7402细胞Bcl-2基因水平显著低于、而Bax基因水平显著高于未转染BEL7402细胞和pU6mRFP Scramble-siRNA转染组BEL7402细胞(0.28±0.15vs0.96±0.21,1.03±0.24,P<0.05;1.09±0.21vs0.26±0.10,0.25±0.07,P<0.01)。结论:siRNA沉默RhoC基因可诱导人肝癌BEL7402细胞凋亡,其机制与下调Bcl-2基因、上调Bax基因表达有关。
Objective: To study the effect and mechanism of siRNA silencing RhoC gene expression on apoptosis of human hepatocellular carcinoma cell line BEL7402, and provide experimental evidence for gene therapy of hepatocellular carcinoma. Methods: The eukaryotic expression vector pU6mRFPRhoC-siRNA of RhoC-siRNA was constructed and transfected into BEL7402 cells. The transfection efficiency was detected by laser confocal microscopy. The silencing effect of RhoC gene was identified by RT-PCR and Western blotting. Flow cytometry, agarose gel electrophoresis The apoptosis of BEL7402 cells was detected by Wright staining. The expression of apoptosis-related genes Bcl-2 and Bax were detected by RT-PCR. RESULTS: The recombinant plasmid pU6mRFPRhoC-siRNA was constructed successfully and the efficiency of transfecting BEL7402 cells was 70%. The efficiency of RhoC gene silencing by RT-PCR and Western blotting was 85% and 82%, respectively. The apoptosis in BEL7402 cells transfected with pU6mRFPRhoC-siRNA was significantly higher than that in BEL7402 cells transfected with pU6mRFP Scramble-siRNA and [(21.00 ± 2.23)% vs (6.47 ± 1.64)% vs (4.63 ± 0.47)%, P <0.01). The DNA of BEL7402 cells in pU6mRFPRhoC-siRNA group showed a typical “ladder” band. Wright’s staining showed that a large number of apoptotic cells were found in BEL7402 cells transfected with pU6mRFPRhoC-siRNA. The Bcl-2 gene level in BEL7402 cells transfected with pU6mRFPRhoC-siRNA was significantly lower than that in BEL7402 cells transfected with pU6mRFPRhoC-siRNA (0.28 ± 0.15 vs 0.96 ± 0.21, 1.03 ± 0.24, P <0.05; 1.09 ± 0.21 vs 0.26 ± 0.10, 0.25 ± 0.07, P <0.01). Conclusion: Silencing RhoC by siRNA can induce apoptosis of human hepatocellular carcinoma BEL7402 cells by down-regulating Bcl-2 gene and up-regulating Bax gene expression.