| Objective:In a preliminary experiment,we found that resveratrol could down-regulate the expression of MARCH 1 in HepG2 and Hep3B cells,therefore,this paper aims to explore the molecular mechanism of resveratrol inhibiting the development of HCC by regulating MARCH1.Methods:In vitro:HepG2 and Hep3B cells were treated with resveratrol at a certain concentration(0,20,40,80 μM)for 48 h.The transcriptional level and protein level of MARCH 1 were detected by real-time quantitative PCR and western blot.The CCK8 and cell colony test were used to detect the effect of resveratrol on the proliferation in HepG2 and Hep3B cells.AnnexinV-FITC/PI double staining method was used to detect the effect on its apoptosis.HepG2 and Hep3B cells were treated with resveratrol(0,10,20,40μM)for 24h,the cell scratch test were used to detect the migration.HepG2 and Hep3B cells were inoculated in the upper chamber and stained after 24 hours.We could calculate the number of cells crossing the membrane,and the percentage of migration and invasion.HepG2 and Hep3B cells were treated with resveratrol at a certain concentration(0,40,80 μM)for 48 h.MARCH 1,PTEN,AKT,P-AKT,STAT3,P-STAT3,VEGF,and Bcl-2 were detected by western blot.The expression levels of MARCH1,PTEN,P-AKT and their mutual regulation mechanism was detected after MARCH1 overexpressing plasmid,MARCH 1-siRNA,PTEN inhibitor BPV(phen),ATK inhibitor(MK2206)acting on HepG2 cells.In vivo:the 4-week-old mice were kept in SPF(no special pathogens)for one week,1×107 HepG2 cell suspension was inoculated on the back of nude mice building xenograft model.After tumor formation,nude mice were randomly divided into three groups,solvent(filtered corn oil)treatment group,50mg/kg resveratrol treatment group,and 100mg/kg resveratrol treatment group.During treatment we would observe the mental state of the nude mice,and record growth of transplanted tumors in nude mice.The treatment lasts for 5 weeks.Nude mice were scanned with magnetic resonance T1 weighted images,T2 weighted images,and coronal T2 weighted images.To observe the pathomorphological changes of tumor tissue so the slices were stainied by HE;the expression of MARCH1 and Ki67 were detected by Immunohistochemistry;the expression of MARCH 1、PTEN、AKT P-STAT3,VEGF,Bcl-2 in tumor tissues were detected by western blot.Results:In vitro:the expression level of MARCH1 were decreased after treated with resveratrol and showed a certain concentration dependence,but the transcription level of MARCH 1 increased slightly.The MG 132 combined with or without resveratrol to treat HepG2 cells,the results showed that MG 132 combined resveratrol reversed the down-regulated expression level of MARCH 1 by resveratrol.Transfecting HepG2 cells with empty vector and MARCH 1 overexpression plasmids,and then co-incubation with or without resveratrol for 24h.The results showed that the number of cells in the MARCH 1 overexpression plasmid with resveratrol combination group was larger than that resveratrol and empty vector combination treatment group.Detecting the expression level of MARCH1 by western blot,which is consistent with the cell phenotype.Resveratrol inhibited the proliferation of HepG2 and Hep3B cells,and the IC50 values of HepG2 and Hep3B cells were 32.33 μM and 113.5μM respectively for 48h.Resveratrol(80μM)significantly inhibited the formation of cell colonies and the size of the cell colonies was significantly smaller than that of the non-medicated group and the low-concentration drug group.Resveratrol promotes the apoptosis of HepG2 and Hep3B cells.The apoptosis rate of HepG2 and Hep3B reached 40%and 51.1%after 80 μM resveratrol treatment,and showed concentration dependence.The results of cell scratch test showed the cell migration rate gradually decreased with the increase of the concentration of resveratrol,under the most significant concentration(40 μM),and the migration rates of HepG2 and Hep3B cells decreased 60%and 56%respectively.Transwell results showed the migration rate of HepG2 cells decreased to 14%and invasion rate decreased to 7.25%,the migration rate of Hep3B cells decreased to 18%and the invasion rate decreased by 19.5%compared with the control groups.Western blot showed that the expression level of MARCH 1,P-AKT/AKT,P-STAT3/STAT3,VEGF,and Bcl-2 decreased,and the expression level of PTEN increased with resveratrol treatment.The level of PTEN increased and P-AKT decreased in HepG2 cells after treated with MARCH1 siRNA;the level of PTEN increased and P-AKT decreased in HL7702 cells after treated with MARCH1 overexpress plasmid;The expression levels of MARCH 1 and P-AKT in the resveratrol combined with BPV(phen)or MK2206 group were the lowest,and the expression of PTEN was the highest.In vivo:compared with the solvent treatment group,the volume and weight of the transplanted tumor in the resveratrol treatment group were significantly reduced,and there was no significant difference in the body weight of nude mice.At the same time,the 7.0T magnetic resonance showed that the volume of the transplanted tumor in the resveratrol treatment group was significantly reduced.HE staining showed loose cell structure and increased apoptotic bodies in the resveratrol-treated group;Immunohistochemical results showed that the expression of MARCH1 and Ki67 was significantly reduced in the resveratrol-treated group.The result showed that the levels of MARCH1,P-AKT/AKT,P-STAT3/STAT3,VEGF,Bcl-2 decreased,but PTEN increased in tumor tissues were detected by western blot.Conclusion:Resveratrol could reduce the expression level of MARCH 1 and regulate the PTEN/AKT signaling pathway,thereby inhibiting the proliferation,migration,invasion and promoting the apoptosis of HepG2 and Hep3B cells.It is suggested that MARCH1 may be a potential molecular target for resveratrol in the treatment of hepatocellular carcinoma,which also prompted us to explore the development of new drugs with MARCH 1 as the target. |
