HBV DNA Polymerase Drives The Glucose Metabolism Reprogramming To Enhance Malignancy In Liver Cancer

[Objective] Hepatocellar carcinoma(HCC)is the sixth common malignancy worldwide and ranks as the fourth leading cause of cancer-related deaths.Chronic hepatitis B virus(HBV)infection is a major risk factor for HCC.Recently,the reprogramming of cancer metabolism has been identified as a hallmark of cancer.The shift from the oxidative phosphorylation metabolic pathway to the glycolysis pathway in HCC meets the demands of rapid cell proliferation and offers a favorable microenvironment for tumor progression.HBV-DNA-Polymerase(HBV-DNA-Pol)functions as a well-known specialized reverse transcriptase(RT)and DNA polymerase to regulate HBV replication,innate immunity and so on.Emerging evidence indicates that HBV-DNA-Pol play a pivotal role in hepatoma cell growth,however,it is not clear whether HBV-DNA-Pol affects the malignant behavior of HCC by influencing the glucose metabolism of HCC.In our previous study,a large number of confirmatory experiments was showed HBV-DNA-Pol could promote the malignancy of hepatocellular carcinoma cells.Co-immunoprecitation,silver staining and mass spectrometry screened proteins that might interact with HBV-DNA-Pol.In this study,we aim to investigate the effect of HBV-DNA-Pol on the occurrence and development of HCC.[Methods] Firstly,we validated the early results of HBV-DNA-Pol,then we established the nude-mouse tumor model to study the effect of HBV-DNA-Pol on the growth and metastasis of HCC in vivo.Secondly,after screening proteins that might interact with HBV-DNA-Pol,we identified PYGL as the research object.The interaction and subcellular localization between HBV-DNA-Pol and PYGL were confirmed by Co-IP and the image analysis.Next,we performed Co-IP assay and used three HBV-DNA-Pol-deleted plasmids constructed earlier to determine the domain of HBV-DNA-Pol binding to PYGL.RT-q PCR,Western blot and immunohistochemistry were used to detect the effect of HBV-DNA-Pol on PYGL expression.To exclude the effects of other HBV proteins on PYGL expression,the effects of HBs Ag,HBc Ag and HBx on PYGL m RNA and protein levels were detected by RT-q PCR and Western blot.After determining the degradation pathway of PYGL,we investigated the potential mechanism which were responsible for regulating PYGL expression by HBV-DNA-Pol via cycloheximide chase assays and protein ubiquitin analysis.Subsequently,quantitative experiments and high-resolution MS etc.were performed to analyze the expression of glycogen and glycolysis-related molecules in HBV-DNA-Pol overexpressed HCC cells.In addition,lactate and glycogen quantitative detection kits were used to detect the changes of lactate and glycogen in xenograft tumors.Finally,the rescue assays demonstrated that the effect of HBV-DNA-Pol on HCC and glucose metabolism was mediated by PYGL.[Results] In vitro and in vivo experiments confirmed that HBV-DNA-Pol promotes malignancy of hepatocellular carcinoma.HBV-DNA-Pol interacts with PYGL and colocalized in the cytoplasm.HBV-DNA-Pol didn't affect its m RNA level but increases PYGL level in HCC cells.TRIM21 is an E3 ubiquitin ligase of PYGL,which can promote the ubiquitination of PYGL and decrease its expression in HCC cells.HBV-DNA-Pol formed a protein complex with PYGL and TRIM21,HBV-DNA-Pol competitively impaired the binding of PYGL to TRIM21 due to its stronger binding affinity to TRIM21,suppressing the ubiquitination of PYGL.HBV-DNA-Pol promoteed glycogenolysis by enhancing the activity of glycogen phosphorylase(PYGL),allowing more G-1-P to enter the glycolysis process,producing more lactate and ATP.In HCC,PYGL acts as an oncogene.The rescue assay demonstrated that knockdown of PYGL could neutralize the promotion effect of HBV-DNA-Pol on the malignant behaviors of liver cancer cells.[Conclusions] In this study,we confirmed HBV-DNA-Pol,TRIM21 interacts with PYGL,and TRIM21 was a E3 ligase of PYGL.HBV-DNA-Pol competitively impaired the binding of PYGL to TRIM21 due to its stronger binding affinity to TRIM21,suppressing the ubiquitination of PYGL and increased PYGL abundance in HCC cells.HBV-DNA-Poly promotes glycogen decomposition by up-regulating PYGL,which leads to the increased flow of glucose into glycolysis,thereby promoting the development of HCC.