Influences Of APE1 Gene Silence On Forming Tumor In Vitro And AP-1 Signal Transduction Pathway Of Human Hepatocellular Carcinoma Cell Line MHCC97-H

Objective to investigate the influences of APE1 gene silence on forming tumor in vitro and AP-1 signal transduction pathway of Human Hepatocellular Carcinoma Cell line MHCC97-H ,which can settle the foundation for the father clarify of the molecular mechanism on proliferation ,invasion and metastasis of human hepatocellular carcinoma(HCC).Methods culture Human Hepatocellular Carcinoma Cell line MHCC97-H . there are three groups:transfected group LV-APE1-shRNA,empty vector transfected group LV-APE1-NC and non-transfected group (control group),then establish an xenograft model of the human hepatocellular carcinoma by respectively inoculated above-mentioned three groups cells . Take the intact subcutaneous tumor tissues and measure their volumes and weight.calulate tumor growth inhibition ratio. Make paraffin section by hematoxylin-eosin staining ,observe the morphology of tumors under light microscope. Then,RT-PCR assay and Western blot assay were performed to detect the mRNA and protein expression levels of APE1gene and genes from AP-1 signal transduction pathway ,which involve c-Fos,c-Jun,caspase-3and MMP-2 .To detection the apoptosis difference among the three groups by TUNEL method,and calculate the apoptosis index of Hepatocarcinoma. Results1.The transplantable tumors of LV-APE1-shRNA group grew slowly, the volume(0.467±0.052)cm3 and weight(0.267±0.082)g were lower cmpared with LV-APE1-NC group (1.700±0.063)cm3,(1.018±0.160)g and control group(1.780±0.095)cm3,(1.107±0.178)g (P<0.01). The tumor growth inhibitory rate was 73.772%.2. necrosis was observed infrequently in LV-APE1-shRNA group by HE dye under light microscope,but observed frequently in LV-APE1-NC group and control group. Apoptosis small body was more compared with the other two groups. Pathologic fission phase five per high power field in LV-APE1-NC group and control group.3.It was shower that the mRNA expression levels of APE1, c-Fos,c-Jun,caspase-3and MMP-2 were lower cmpared with LV-APE1-NC group and control group (P<0.01). After APE1 gene silence , mRNA expression levels of APE1 decreased by 90.622% ,c-Fos decreased by47.580%,c-Jun decreased by 47.402%,caspase-3 decreased by 65.355%,MMP-2 decreased by 64.369%。And their expressions were positive correlation.4.It was showed that the protein expression levels of APE1, c-Fos,c-Jun,caspase-3and MMP-2 were lower cmpared with LV-APE1-NC group and control group (P<0.01). After APE1 gene silence , protein expression levels of APE1 decreased by 72.439% ,c-Fos decreased by71.872%,c-Jun decreased by 80.399%,caspase-3 decreased by 75.616%,MMP-2 decreased by 88.393%。And their expressions were positive correlation.5. It was found that apoptosis count of LV-APE1-shRNA group was higher compared with the other two groups ( P<0.01 ), the apoptosis index of LV-APE1-shRNA group was 5.503%,LV-APE1-NC group was 2.911% and control group was 2.569% by TUNEL method. Results Conclusions1.It was successful to establish an xenogrft model of the Human Hepatocellular Carcinoma Cell line MHCC97-H . APE1 gene silence of MHCC97-H by infected lentivirus vector can inhibit observably the growth of transplant tumor.2.RT-PCR assay and Western blot assay showed that the mRNA and protein expression levels of APE1, c-Fos,c-Jun,caspase-3 and MMP-2 were inhibited observably,and APE1 can regulat expression of c-Fos,c-Jun,caspase-3 and MMP-2 positively. APE1 could have a hand in proliferation ,invasion and metastasis of human hepatocellular carcinoma by AP-1 signal transduction pathway.3.Tumor morphology observation and apoptosis detection assay by TUNEL method showed that APE1 gene silence can suppress proliferation and promote apoptosis of hepatocarcinoma. Conclusions...