Biological Function And Mechanism Of Chronic ER Stress Induced By LHB In The Process Of Hepatocellular Carcinoma

Hepatocellular carcinoma(HCC)is the sixth most common malignancy and the third deadliest cancer in the world,accounting for 8.2% of all tumor-related deaths worldwide.The occurrence of liver cancer is multifactorial,with alcohol,hepatitis B virus(HBV)and aflatoxin as its risk factors.Since there is still no effective clinical drug to clear HBV,chronic HBV infection becomes the most important risk factor.Hepatitis B infected by HBV is the main cause of liver cancer in China,accounting for about 80% according to statistics.More than 2 billion people worldwide were exposed to HBV and nearly 296 million people are chronic HBV carriers by 2022.Gross glassy hepatocytes(GGHs)are often observed in the hepatocytes of clinical HBVinfected patients,which are precancerous cytopathological changes caused by the accumulation of HBV surface proteins in the lumen of the ER due to their massive synthesis and inability to be secreted properly.The hepatitis B virus envelope is mainly composed of HBV surface protein,which has three forms,including large HBV surface protein(LHB).The study found that LHB was persistently and highly expressed in the tumorigenesis,indicating that LHB played an important role in the hepatic tumorigenesis.Sustained hepatocyte proliferation and GGHs accompanied by accumulation of LHB in the ER have been observed in LHB transgenic mice in the development of HCC.However,its molecular mechanism of promoting cancer has not been fully elucidated.Therefore,the mechanisms of LHB-driven HCC progression need to be fully explored.Based on these problems,we explored the pathogenesis of LHB as a potential oncogenic factor in HBV-related HCC progression to provide potential therapeutic options for prevention and intervention.This study mainly includes three parts.Part Ⅰ: LHB-induced ER stress promotes HCC by regulating cell cycleWe first explored the expression of LHB in patients with HBV(+)HCC and found that the expression of LHB in HCC tissues was significantly higher than that in adjacent tissues and the high expression of LHB in tumor tissues predicted a poor survival and prognosis.Overexpression of LHB induced ER stress and activation of the UPR pathway,especially PERK and IRE1α pathways.To further investigate the pathogenic mechanism of LHB,we established LHB overexpression stable cell lines and performed RNA-seq.GSEA analysis showed that LHB overexpression significantly affected the cell cycle of HCC cells.Cell cycle analysis indicated that G1 arrest was reduced and the proportion of cells in the G2 and M phases increased,which further contributed to acceleration of cell proliferation.In order to investigate the link between ER stress induced by LHB and cell cycle,LHB-expressing cells were treated with ER stress inhibitor,4-PBA.The results showed that the transition of G1/S phase was significantly slowed down,and the cell proliferation rate was also slowed down.This demonstrated that LHB as a potential oncogenic factor regulated the cell cycle through inducing ER stress to promote the progression of liver cancer.Part Ⅱ: LHB-induced ER stress transcriptionally regulates p27 expressionWe explored the molecular mechanism of LHB in accelerating cell cycle and found that the levels of cyclins and cyclin-dependent kinases(CDK),which are positive growth regulators of cell cycle,were significantly increased.In contrast,the expression of CDK inhibitors,p27 and p21 was significantly decreased,but their m RNA expression was significantly increased,suggesting that post-translational modification might play a role in the stability of p21 and p27 proteins.Further study showed that LHB as an oncogenic factor promoted hepatocarcinogenesis by accelerating the ubiquitination of p27 and p21 and thus accelerating cell cycle progression.ER stress affects the cell life cycle and determines cell fate.Next,we investigated how ER stress induced by LHB affects cell cycle progression.We found that when ER stress was inhibited in LHB overexpressing cells,the decrease of p27 protein expression was reversed,but the expression of p21 was still down-regulated.These data suggest that LHB-induced ER stress affects cell cycle progression by regulating p27 expression.We then examined whether the transcription factors of downstream of the PERK and IRE1α pathways regulate p27 transcription.Treatment with IRE1α and PERK pathway inhibitors resulted in a significant decrease in p27 m RNA expression.After treatment with inhibitors of IRE1α and PERK pathway,the expression level of p27 m RNA decreased,and the expression of p27 protein decreased more significantly.We further confirmed that p27 was regulated by ATF4 and XBP1 s.LHB overexpression significantly increased the m RNA expression of p27,which was suppressed after silencing ATF4 or XBP1 s.A dual-luciferase reporter assay showed that ATF4 and XBP1 s significantly increased the promoter activity of p27.Ch IP analysis confirmed that ATF4 and XBP1 s could bind to the promoter region of p27 to regulate p27 transcription.These results indicated that LHB-induced ER stress mediated p27 transcription through IRE1α-XBP1 s and PERKATF4 pathways.Under ER stress,the translation of most proteins is suppressed,except for a few proteins that can be translated through internal ribosome entry site(IRES)to maintain essential biological activities.We found that the IRES activity of p27 m RNA 5’-UTR was significantly enhanced under LHB-induced ER stress,which implies that p27 can still be selectively translated under ER stress.Part Ⅲ: LHB-induced ER stress regulates post-translational modification of p27 mediating cell cycle progression during HCC progressionER stress usually leads to activation of the ERAD pathway.ERAD is a protein quality control system that is evolved to maintain ER homeostasis in cells.HRD1 is an important part of ERAD,the protein and m RNA expression of HRD1 increased significantly when LHB was overexpressed.After the overexpression of LHB,the protein expression of p27 increased significantly with HRD1 silencing.Cell cycle analysis showed that G1/S arrest increased after HRD1 silencing.Co-immunoprecipitation assays showed that HRD1 could interact with p27.In addition,knockdown of HRD1 in LHB overexpressing cells significantly reduced the ubiquitination level of p27.Moreover,the ubiquitination of p27 was obviously enhanced when LHB was overexpressed,which was reduced by silencing HRD1.Immunohistochemical staining showed that the expression levels of HRD1 and p27 were negatively correlated in patients with HBV(+)HCC.Collectively,these results indicated that HRD1 was an E3 ubiquitin ligase of p27 that accelerated the ubiquitination and degradation of p27.In summary,the accumulation of LHB in the ER induced ER stress,and the activated UPR pathway transcriptionally regulated the m RNA expression of p27,subsequently p27 was selectively translated in an IRES-dependent manner,finally the ERAD-related E3 ligase HRD1 was involved in the ubiquitinated degradation of p27 protein,which ultimately led to the acceleration of cell cycle progression.This study elucidates the mechanism by which LHB as a potential oncogenic factor regulates the transcriptional and post-translational modifications of p27 through inducing ER stress,thus achieving precise regulation of p27 and ultimately promoting tumor progression.