| Hepatitis B virus(HBV)is closely related to the carcinogenesis of HBV-related hepatocellular carcinoma(HCC).HBV and its multiple viral proteins can promote the invasion,metastasis and angiogenesis of HCC through various pathways.HBV surface antigen(HBs Ag)is the most abundant viral protein,which contains three forms,i.e.,large HBs Ag(LHBs),medium HBs Ag(MHBs)and small HBs Ag(SHBs).Synthesizing and processing of a large number of SHBs in endoplasmic reticulum(ER)result in ER stress,and will lead to the change of downstream signal pathway.The clinical study showed that the occurrence risk of HCC with serum SHBs > 1000 IU / ml increased by3.86 times,and in mice,the elimination of SHBs with specific antibody can greatly reduce the probability of HCC,suggesting that SHBs is closely related to the occurrence and development of HCC.Therefore,this paper aims to study the effect and mechanism of SHBs on metastasis and angiogenesis of liver cancer through ER stress.The first part of this study is to investigate the effect of SHBs on metastasis and angiogenesis of HCC.First,hepatoma cell lines Huh7-SHBs and Hep G2-SHBs stably expressing SHBs-Flag fusion protein(control cells were Huh7-Flag and Hep G2-Flag respectively)were generated.Subsequently,through scratch test and Transwell chamber assay(with or without matrix glue),it was found that SHBs could promote the migration and invasion of hepatoma cells;by orthotopic implantation of hepatoma in nude mice,it was found that SHBs could promote the intrahepatic and pulmonary metastasis of hepatoma cells and shorten the survival period of nude mice.In addition,it was found that the culture supernatant of hepatoma cells expressing SHBs could promote the migration and ring forming of human umbilical vein endothelial cells(HUVECs).Nude mice subcutaneous tumorigenesis and immunohistological examination of transplantation tumor demonstrated that SHBs could promote tumor growth and increase tumor microvascular density(MVD).Furthermore,statistical analysis of clinical data and pathological feathers in 112 cases of liver cancer tissues revealed that HBs Ag was positively related to cirrhosis,AFP,tumor number,encapsulates and TNM stage,and the total survival period of HBs Ag positive patients was shorter than that of HBs Ag negative patients,HBs Ag was an independent risk factor for poor prognosis of liver cancer.Immunohistochemistry staining confirmed the positive correlation between HBs Ag and microvessel density.Taken togather,these results indicate that SHBs can promote the metastasis and angiogenesis of HCC.The second part of this study aims to explore the mechanism of SHBs in promoting metastasis of HCCThe first chapter studied the effect of SHBs on the epithelial-mesenchymal transition(EMT)of hepatoma cells.By Western blot analysis,real-time PCR or immunohistochemistry staining,we found that SHBs down regulated E-cadherin and up regulated N-cadherin,vimentin and m RNA expression in hepatoma cells,while SHBs down regulated E-cadherin and up regulated N-cadherin in mice transplantation tumor tissues and tumor tissues from HCC patients.Western blot analysis,real-time PCR and luciferase assay were used to detect transcriptional and translational level of EMT related transcription factors.The results revealed that SHBs enhanced the promoter activitied and expression of snail,slug,Twist1,and ZEB1 in m RNA and protein level,suggesting that SHBs could promote EMT process of hepatoma cells.The second chapter studied the mechanism of SHBs in regulating EMT.SHBs expressing hepatoma cells were treated with various EMT-related pathway Inhibitors,and the results showed that only S3I-201,the inhibitor of STAT3,reversed the effect of SHBs on E-cadherin expression and cell migration,suggesting that SHBs might promote EMT and cell migration through Stat3.To confirm further,two specific si RNAs and inhibitors targeting STAT3 were used to treat SHBs expressing hepatoma cells.Western blot,real-time PCR and Transwell chamber assay showed that si RNA and inhibitors could reverse the effect of SHBs on EMT related proteins at protein and m RNA levels,and inhibit the ability of SHBs in promoting cell migration and invasion.In addition,orthotopic liver transplantation in nude mice using STAT3-knockdown SHBs expressing hepatoma cells showed the reduced tumor metastasis.These results indicated that SHBs can EMT and metastasis of HCC through STAT3 pathway.In the third chapter,the phosphorylation of STAT3(Tyr705)by SHBs through JAK2 was studied.Western blot analysis showed that SHBs promoted the phosphorylation of STAT3 tyr705,but had no effect on ser727.In addition to the cytoplasm,SHBs also increased the phosphorylation level of STAT3(tyr705)in the nucleus,as confirmed by nuclear/cytoplasmic separation experiment and immunofluorescence staining.Immunohistochemistry staining and Western blot analysis also verified that SHBs increased the phosphorylation of STAT3(Tyr705)in mice transplantation tumor tissues and tumor tissues from HCC patients.Western blot analysis showed that SHBs promoted the phosphorylation of JAK2 tyr1007/1008 site,and immunoprecipitation(IP)showed that SHBs did not interact with STAT3 and JAK2.Furthermore,Western blot analysis showed that the activation of JAK2/STAT3 by SHBs was not related to SOCSs and SHP1/2 family.The results also showed that AG490 could inhibit the effect of SHBs on STAT3 and EMT related proteins at protein and m RNA levels,and inhibit the ability of SHBs in promoting cell migration and invasion.Taken together,the all these results showed that SHBs enhanced STAT3 phosphorylation by promoting JAK2 phosphorylation,and finally promoted EMT and migration and invasion of hepatoma cells.In the fourth chapter,the mechanism of SHBs in promoting JAK2/STAT3 pathway was investigated.By cytokines array screening,fibroblast growth factor-19(FGF19)was found to be upregulated by SHBs,it was further verified by Western blot,real time PCR and ELISA,which showed that SHBs enhanced the expression of FGF19 both at m RNA and protein levels,but had no effect on its receptor FGFR4.Western blot and Transwell chamber assay showed that knockdown of FGF19 or FGFR4 by specific si RNAs inhibited the ability of SHBs in promoting the phosphorylation of JAK2 and STAT3 and the migration and invasion of hepatoma cells,which indicating that SHBs promoted the metastasis of HCC through enhancing the expression of FGF19.To investigate the role of ER stress in SHBs pathogenicity,ER stress alleviators 4-PBA and TUDCA,as well as SHBs S204 R mutant were applied,and the Western blot analysis revealed that 4-PBA and TUDCA could inhibit,while the SHBs S204 R mutant enhanced the effects of SHBs on the FGR19 expression.Taken together,these results indicated that SHBs promoted the expression of FGF19 through ER stress,which sequentially activate JAK2/STAT pathway,and ultimately promote the migration and invasion of hepatoma cells.The third part of this study aims to explore the mechanism of SHBs in promoting angiogenesis of HCC.The first chapter studied the mechanism of SHBs in promoting angiogenesis.By Western blot,real-time PCR and ELISA,it was found that SHBs enhanced the expression of vascular endothelial growth factor A(VEGFA).Further experiments showed that the mutation of SHBs initiation code in HBV weakened the effect of1.2-fold HBV on the expression of VEGFA.When the culture supernatant were obtained from SHBs expressing cells treated with two si RNA targeting VEGFA,and used to culture HUVECs cells,the abilities of migration and ring forming of HUVECs cells were.These results suggest that SHBs can enhance the expression of VEGFA and promote the angiogenesis of HCC.The second chapter studied the mechanism of SHBs in enhancing the expression of VEGFA.Western blot,ring forming of HUVECs and Transwell chamber assay showed that ER stress inhibitor 4-PBA and TUDCA could inhibit the expression enhancement of VEGFA by SHBs,and the expression of VEGFA was further enhanced by SHBs G154 R mutant,indicating that SHBs enhanced the expression of VEGFA through ER stress.In order to further study the effect of SHBs on the ER stress-induced pathway changes,si RNAs targeting IRE1,PERK or ATF6,which were the three main downstream pathways of ER stress,were used to treat SHBs expressing hepatoma cells.Western blot,ring forming of HUVECs and Transwell chamber assay showed that si RNAs knockdown of these three pathways could inhibit the effect of SHBs on the enhancement of VEGFA,as well as the ability of ring forming and migration of HUVECs.These results indicated that SHBs can enhance the expression of VEGFA through downstream pathways of ER stress,i.e.,IRE1-XBP1,PERK-ATF4 and ATF6,and promote angiogenesis. |
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