Wild Type HBsAg Inhibits Hepatocellular Carcinoma Through Competitive Blocking Of KPNA2 Nuclear Transport

Objective:This study aims to explore the inhibition of Hepatitis B surface Antigen(HBsAg)to KPNA2 nuclear transfer function and research the inhibition to Hepatocellular Carcinoma(HCC)at the moleular and cellular levels.Method:1.Testing KPNA2 mRNA and protein expression levels by RT-qPCR and Western Blot in LO2 cells and SMMC 7721 cells,respectively.2.Using the recombinant plasmid pIRES2-HBsAg existing in the laboratory as the template,the twin-PCR primers were designed to obtain the HBsAg fragment,then the fragment was inserted into the pIRES2-EGFP.Using the cDNA of SMMC7721 cells as the template,the cloned primers were designed according to the KPNA2 gene sequence founded in GenBank(NM-002266),and the KPNA2 fragment was inserted into the above plasmid to be constructed as pIRES2-HBsAg-KPNA2 coexpression vector.3.After treating cells with different concentrations of ivermectin,the difference of E2F1 expression was detected by nuclear separation experiment,and the optimum treatment concentration was obtained.4.Control and p-HBsAg plasmid were transfected to SMMC7721 cells respectively,and immune co-cipitation verified whether HBsAg was combined with KPNA2 in SMMC7721 cells.5.Control,p-HBsAg,p-KPNA2,p-HBsAg-KPNA2 plasmids were respectively transferred to SMMC7721 cells,and Ivermectin was used to treat SMMC7721 cells alone.Then detecting the expression levels of E2F1,c-Myc,PLAG1 and STAT3 protein in internal and external nuclei through nuclear separation experiments.6.Control,p-HBsAg,p-KPNA2,p-HBsAg-KPNA2 plasmids were respectively transferred to SMMC7721 cells,and Ivermectin was used to treat SMMC7721 cells alone.Then detecting PDGF,AKt,F0XO3 a,IGFII,cyclinD1 mRNA expression differences by RT-qPCR.7.Control,p-HBsAg,p-KPNA2,p-HBsAg-KPNA2 plasmids were respectively transferred to SMMC7721 cells,and Ivermectin was used to treat SMMC7721 cells alone.Then detecting the absorbance of SMMC7721 cells at OD450 nm after 24 h,48h,72 h by CCK8 experiment,and detecting the proliferation of SMMC7721 cells by EDU imaging experiment in 48 h after transfection.8.Control,p-HBsAg,p-KPNA2,p-HBsAg-KPNA2 plasmids were respectively transferred to SMMC7721 cells,and Ivermectin was used to treat SMMC7721 cells alone.Then detecting and comparing the motor ability of cells through cell scratches,migration and invasion experiments.Results:1.KPNA2 mRNA and protein expression levels in SMMC 7721 cells were significantly higher than that in LO2 cells.2.The enzyme-cut product of recombinant plasmid was detected by 1% agarose gel electrophoresis,and it was found that the stripe position was consistent with the expectation,and the sequencing results of the biological company were in accordance with the experimental design.3.Control and p-HBsAg plasmid were transfected to SMMC7721 cells respectively,and immune co-cipitation confirmed that HBsAg combined with KPNA2 in SMMC7721 cells.4.The inhibition capacity to nuclear transport of KPNA2 geted to its climax when the concentration of ivermectin was 5uM.5.Control,p-HBsAg,p-KPNA2,p-HBsAg-KPNA2 plasmids were respectively transferred to SMMC7721 cells,and Ivermectin was used to treat SMMC7721 cells alone.The results of nuclear separation showed that the expression level of E2F1,cMyc,PLAG1 protein nucleus in p-HBsAg and ivermectin groups decreased significantly compared with that of Control,p-HBsAg-KPNA2,p-KPNA2 groups.The expression level of STAT3 protein nucleus in p-KPNA2 group and p-HBsAgKPNA2 group were significantly higher than that in control,p-HBsAg and ivermectin groups.6.Control,p-HBsAg,p-KPNA2,p-HBsAg-KPNA2 plasmids were respectively transferred to SMMC7721 cells,and Ivermectin was used to treat SMMC7721 cells alone.The results of RT-qPCR experiment showed that the expression levels of PDGF,AKt,IGFII mRNA in p-HBsAg group and ivermectin group decreased remarkably compared with that of control,p-HBsAg-KPNA2,p-KPNA2 groups,while Foxo3 a mRNA level increased.The level of cyclinD1 mRNA in control group was much higher than that of Ivermectin and HBsAg groups,but lower than that of pKPNA2 and p-KPNA2-HBsAg groups.7.Control,p-HBsAg,p-KPNA2,p-HBsAg-KPNA2 plasmids were respectively transferred to SMMC7721 cells,and Ivermectin was used to treat SMMC7721 cells alone.The absorbance of the control,p-HBsAg-KPNA2 and p-KPNA2 groups at OD450 nm continued to increase with the prolongation of incubation time.The DNA replication ability of p-HBsAg and Ivermectin groups were lower than that of control,p-HBsAg-KPNA2 and p-KPNA2 groups.8.Control,p-HBsAg,p-KPNA2,p-HBsAg-KPNA2 plasmids were respectively transferred to SMMC7721 cells,and Ivermectin was used to treat SMMC7721 cells alone.The scratches area of p-HBsAg and Ivermectin group was obviously larger than that in control,p-HBsAg-KPNA2 and p-KPNA2 groups,and the cell migration and invasion experiments showed that the number of cells migrating and invading in p-HBsAg,ivermectin groups was much lower than that in control,p-HBsAg-KPNA2 and p-KPNA2 groups.Conclusion:HBsAg inhibits E2F1,c-Myc,PLAG1 transport into the nucleus by binding KPNA2,thereby inhibiting PDGF,akt,IGFII,cyclin D1 expression,increasing Foxo3 a expression,and inhibiting hepatocellular carcinoma.