Study On The Role Of GCF2 In Migration And Invasion Of Hepatical Carcinoma

Objective:the present study was aimed to investigate the role of GCF2 in hepatocellular carcinoma(HCC)cell migration and invasion,and to identify target genes regulated by transcriptional factor GCF2 involved in HCC cell migration and metastasis.These results would provide the foundation for explicating molecular mechanism of HCC metastasis.Methods:(1)Three HCC cells were included in this study:HepG2 and BEL-7404 which showed high expression of GCF2,and high transfer HCC cell line MHCC97-H.GCF2 expression were silenced by RNA interference(RNAi)in these three HCC cell lines.And transwell migration and invasion assay were performed to detect the effect of GCF2 silence on HCC cell lines migration and invasion.(2)Stable silence GCF2 HCC cell lines were constructed by lentiviral shRNA vector system which suppressed GCF2 expression via RNAi.Then the effect of silence GCF2 on metastasis of HCC cell lines in vivo were determined by planting silence GCF2 HCC cell lines in situ in nude mice model.(3)GCF2 expression were silenced by RNA interference(RNAi)in HepG2 and BEL-7404 which showed high expression of GCF2,real-time fluorescent quantitative PCR and western blot were performed to detect the expression of 13 potential target that regulated by GCF2 in cell migration and invasion which sift out by the early stage of the research study.(4)To further determine transcriptional regulation of GCF2 on key target gene,dual luciferase report system tests were performed to analyze promoter activity of the key target genes.Results:(1)GCF2 gene silencing can significantly suppress migration and invasion ability of the hepatocellular carcinoma cell line HepG2,BEL-7404 and MHCC97-H.In all of three kinds of HCC cell lines,the cells number of siRNA-GCF2 group traversed the cell-permeable membrane with or without martrigel were significantly less than the number of siRNA-NC group.And the result of the independent sample t-test was statistically significant difference(P<0.01).(2)After implantation of the wild type cell group,LV3-NC group and GCF-shRNA group in nude mouse liver in situ respectively,the metastatic ability of each group showed significantly difference:the intrahepatic metastasis number of GCF-shRNA group were decreased compared with wild type cell group and LV3-NC group(P<0.01).Furthermore,silence GCF2 has no effect on pancreas lung metastasis,gastric metastasis,lung metastasis of HCC cell in vivo(P>0.05).(3)real-time fluorescent quantitative PCR and western blot results showed that Galectin-1 expression were both increased in HepG2 and BEL-7404 when silencing GCF2,but MYD88D expression were suppressed in HepG2,increased in BEL-7404.Dual luciferase report system tests results confirmed that GCF2 had a negative role in regulating Galectin-1 gene promoter activity that is associated with cell invasion and migration.Conclusion:Our results revealed that GCF2 promoted HCC cell migration and invasion,preliminary findings that when silenced GCF2 in BEL-7404 can suppressed intrahepatic metastasis,but no effect on pancreas lung metastasis,gastric metastasis,lung metastasis.GCF2 have Different adjustment to the downstream target genes which related with Cell migration.GCF2 can suppressing the transcriptional expression of downstream target genes Galectin-1.but the detail mechanism of GCF2 regulate MyD88 to effect Liver cancer cell migration need more discussion.