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siRNA silencing RhoC expression induces apoptosis of human hepatocellular carcinoma BEL7402 cells
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Abstract:
Objective:To study the effect of siRNA silencing RhoC expression on apoptosis in human hepatocellular carcinoma BEL7402 cells and the related mechanism, so as to provide an experimental evidence for liver cancer gene therapy. Methods: RhoC-siRNA eukaryotic vector pU6mRFP RhoC-siRNA was constructed and transfected into BEL7402 cells, and the transfection efficiency was examined by confocal microscope. RT-PCR and Western blotting analysis were conducted to identify the effect of RhoC gene silencing; flow cytometry, agarose gel electrophoresis and Wright staining were applied to detect apoptosis of BEL7402 cells; and the expression levels of apoptosis related genes were determined by RT-PCR. Results: The pU6mRFP RhoC-siRNA recombinant plasmid was successfully constructed, and its transfection efficiency in BEL7402 cells was 70%. RT-PCR and Western blotting analysis results showed that the silencing rate of RhoC were 85% and 82%, respectively. The apoptosis rate of BEL7402 cells in pU6mRFP RhoC-siRNA transfected group was significantly higher than those in untransfected and pU6mRFP Scramble-siRNA transfected groups (\[21.00±2.23\]% vs \[6.47±164\]％,\[4.63±0.47\]%，P<0.01). Typical apoptotic DNA “ladder” appeared in pU6mRFP RhoC-siRNA transfected BEL7402 cells in gel electrophoresis analysis, and Wright staining showed a lot of apoptotic BEL7402 cells in pU6mRFP RhoC-siRNA group. Compared with untransfected and pU6mRFP Scramble-siRNA transfected BEL7402 cells, Bcl-2 gene expression in pU6mRFP RhoC-siRNA group was significantly decreased and Bax gene expression was significantly increased (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±007, P<0.01). Conclusion: siRNA silencing RhoC gene expression can induce apoptosis in human hepatocellular carcinoma BEL7402 cells, which may be related to the down-regulated expression of Bcl-2 gene and up-regulated expression of Bax.
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