Upregulation Of Gap Junction Protein α1(GJA1) Inhibits HCC Progression By Regulating RALA Activating Hippo Signaling Pathway

Research purpose:In this study,cellular and molecular biology and animal experiments were used to explore and verify how gap junction protein α1(GJA1)mediated RALA to activate the signal transduction of Hippo tumor suppressor pathway,thus affecting the progression of human hepatocellular carcinoma(HCC).We hope to further clarify the biological effect of GJA1 on HCC and the potential developmental mechanism through experimental studies.Research methods:In the previous studies of this subject,we have confirmed that there are significant differences in GJA1 expression in HCC tissues,as well as the relationship between GJA1 and survival prognosis.We also identified differentially expressed genes(DEGs)using bioinformatics and analyzed their function and pathway enrichment,suggesting that RALA is a downstream target of GJA1.On the basis of previous studies,we again analyzed the expression difference of GJA1 in HCC tumor tissues and paracancerous tissues by real-time quantitative fluorescence PCR(q RT-PCR)and Western Blot(WB).In vitro,GJA1 expression was overexpressed in Hep3 B cells and knocked down in PLC/PRF/5 cells by si RNA transfection.Through CCK8 experiments,the effects of GJA1 on proliferation activity of Hep3 B and PLC/PRF/5 cells were investigated;The effect of GJA1 on the migration ability of Hep3 B and PLC/PRF/5 cells was detected by scratch test;Transwell and Invasion experiments were used to detect the effects of GJA1 on the migration and invasion activities of Hep3 B and PLC/PRF/5 cells.In order to explore and verify the molecular mechanism of whether GJA1 influences HCC through regulation of RALA,we used Western Blot to explore the regulatory relationship between GJA1,RALA and Hippo signaling pathways.In order to explore and verify the molecular mechanism of whether GJA1 influences HCC through regulation of RALA,we used Western Blot to explore the regulatory relationship between GJA1,RALA and Hippo signaling pathways.Furthermore,in vitro cell biology experiments were conducted to verify how GJA1 mediated RALA to activate the signal transduction of the Hippo tumor suppressor pathway,thus affecting the progression of HCC cells.In addition,Hep3 B cell lines stably expressing GJA1 were constructed by lentivirus,and three groups of nude mouse models were established by subcutaneous injection,including stably expressing GJA1 group(LV-GJA1 group),negative control group(LV-Con group),and co-transfection group LV-(GJA1+RALA).The growth of mice was recorded regularly during feeding period,and tumors were taken out 30 days later for subsequent experiments to further demonstrate how GJA1 mediated RALA to activate the signal transduction of Hippo tumor suppression pathway,thus affecting the progression of HCC.Results:(1)GJA1 expression was down-regulated in HCC carcinomas compared with paracancerous tissues;The expression of GJA1 in HCC cell lines(Hep3B、HCCLM3、Huh7 and PLC/PRF/5)was lower than that in normal hepatic cell(L02).(2)Compared with the control group,the proliferation activity,migration and invasion ability of Hep3 B cells in the transfected plasmid overexpressing GJA1(pc DNA-GJA1)group were significantly decreased,while the proliferation activity,migration and invasion ability of PLC/PRF/5 cells in the knockdown GJA1(si-GJA1)group were significantly increased.(3)The overexpression of GJA1 in Hep3 B cells down-regulated the protein expression level of RALA,while the knockout of GJA1 in PLC/PRF/5 cells up-regulated the protein expression level of RALA,and their expressions were negatively correlated.In addition,TCGA database analysis showed that RALA expression was significantly higher in HCC tumor tissues than in paracancerous tissues,and patients in the group with high RALA expression had a worse prognosis than patients in the group with low RALA expression.(4)RALA can reverse the effect of GJA1 on HCC cell proliferation,migration and invasion through in vitro cell experiments.(5)After the overexpression of GJA1 in Hep3 B cells,the expression of RALA protein decreased,and the phosphorylation levels of LATS1 and YAP in the Hippo signaling pathway increased.After GJA1 silencing in PLC/PRF/5 cells,RALA protein expression was increased and phosphorylation of LATS1 and YAP was decreased.(6)The results showed that high expression of GJA1 could inhibit tumor growth in nude mice,and its effect could be reversed by RALA.Conclusion:The up-regulated expression of GJA1 activates the internal signal transduction of the Hippo tumor suppressor signaling pathway by regulating the expression of the downstream protein RALA,promotes the increase of the phosphorylation levels of LATS1 and YAP,and ultimately prevents the entry of YAP into the nuclear transcription and translation,thereby inhibiting the progress of HCC cells,such as the ability of proliferation,migration and invasion.