The Effects And Mechanisms Of Cdc25C Down-regulation On HCC Proliferation And Apoptosis In Mouse

Objective:To study the effects of down-regulation of Cdc25C expression on proliferation,migration and apoptosis of hepatocellular carcinoma cells by silencing Cdc25C gene by siRNA interference technique,and to explore the mechanism of Cdc25C in the development of hepatocellular carcinoma.Methods:(1)the mouse hepatoma Hepa 1-6 cell lines with Cdc25C expression was detected by RT-PCR assay.(2)The specific Cdc25C interference sequence(siRNA)with labeling FAM was transfected into Hepa 1-6 cells to construct Cdc25C down-expression hepatocytes.The interference experiments were divided into blank control group(untransfected),negative control group(siRNA plasmid empty vector)and experimental group(siRNA-Cdc25C-1,siRNA-Cdc25C-2 and siRNA-Cdc25C-3 interference sequence).After transient transfection,Fluorescence microscopy and RT-PCR were used to screen the best transfection conditions.The Cdc25C mRNA and protein expression in Hepal-6 cells before and after transfection were detected by qPCR and Western blot methods,respectively.The best interference sequence was determined.(3)The effects of Cdc25C down-regulation on the proliferation and migration of Hepal-6 cells were observed by CCK-8 assay and wound healing assay.(4)The effects of Cdc25C down-regulation on cell cycle and apoptosis of Hepal-6 cells were detected by flow cytometry.(5)The expression of GRP78(the major inducer of endoplasmic reticulum stress response),XBP-1(the major inducer of unfolded protein response),and CHOP(the specific transcription factor of endoplasmic reticulum stress)in hepatocytes were detected by qPCR method.Results:(1)The results of agarose gel electrophoresis showed that theCdc25C gene was positive in Hepal-6 cells and the result of gene sequencing showed the gene was correct.(2)The transfection efficiency of siRNA+Lipo2000 solution with the concentration of "30+1.5" was the highest,and the optimum interference time was 24 h.The expression of Cdc25C gene and protein in the experimental group was significantly lower than that in the control group,and the lowest expression was found in the siRNA-Cdc25C-1 group(P<0.01).(3)After reducing the expression of Cdc25C,it was found that the survival rate of the experimental group cells was significantly lower than that of the control group.There was significant difference between the two groups(P<0.05).The results of wound healing assay also showed that the repair rate of cells in the experimental group cells was significantly lower than that in the control group(P<0.05).(4)Cell cycle was detected by flow cytometry,and the expression of Cdc25C in Hepal-6 cells was decreased,S phase cells increased,G2/M phase cells decreased.At the same time,Apoptosis was detected by flow cytometry,the results showed that the apoptosis of Hepal-6 cells significantly increased after interfering the Cdc25C expression.Compared with the control group,the difference was statistically significant(P<0.05).(5)After Cdc25C down-regulation,the expression of GRP78,XBP-1 and CHOP in the experimental group was significantly higher than that in the control group.The difference was statistically significant(P<0.05).Conclusion:Down-regulating the expression of Cdc25C gene can inhibit the proliferation of hepatoma cells and promote the apoptosis of hepatoma cells.The mechanism may be related to the endoplasmic reticulum stress induced by the down-regulation of Cdc25C expression in hepatoma cells.