Study On The Molecular Mechanism Of ATF6 Downstream Gene PPM1H Regulating HCC Progression Through RPS6KB1 And The Role Of ADAR1 And Downstream Genes In Affecting HBV Infectio

Hepatocellular carcinoma(HCC)is the sixth most common cancer in the world and is particularly prevalent in East and Southeast Asia,including China,resulting in approximately 830,000 deaths annually worldwide.Due to the lack of early diagnostic markers and limited therapeutic methods,the clinical therapeutic effect of HCC is not satisfactory,leading to high rate of recurrence and metastasis.The development of HCC involves the regulation of complex signal transduction pathways.Tumor cells are susceptible to intracellular or extracellular stimulation which induce ER Stress and unfolded protein response(UPR).Transcription factor ATF6,as one of the ER stress receptors,is cleaved and activated in Golgi apparatus and transferred into the nucleus.It mediated the transcription and expression of a series of UPR related genes.Previous studies of our laboratory have found that the gene polymorphism of ATF6 is associated with genetic susceptibility to liver cancer.ATF6 promotes the proliferation,migration and invasion of HepG2 cells.Protein phosphatase PPM1H regulates cancer-related signaling pathways such as BMP/SMAD pathway,and is related to tumor suppressor p27 mediated drug resistance.PPM1H plays a regulatory role in colorectal cancer,breast cancer,pancreatic cancer and other cancers.To identify the molecular mechanism by which ATF6 regulates the progression of HCC through down-regulating the expression of PPM1H,and to provide new evidences for the development of targeted therapy strategies for HCC,transmission electron microscopy was used to detect the effect of ATF6 overexpression on the morphology of endoplasmic reticulum.It was found that the volume of the endoplasmic reticulum of ATF6 overexpressed HepG2 cells were expanded.PPM1H was screened and verified as the downstream gene of ATF6 by RNA-seq and qRT-PCR.Western Blot and qRT-PCR were performed to identify that the expression level of PPM1H mRNA and protein were upregulated in the liver tissues of Atf6-knockout mice.Through daul-luciferase reporter assays and mRNA degradation rate assays,it was found that ATF6 had no significant influence on PPM1H promoter activities and mRNA degradation rate.The influences of PPM1H on HCC cells were investigated by MTT proliferation assays,colony formation assays,Transwell migration and invasion assays,and Western Blot detection of epithelial mesenchymal transformation(EMT)markers.The results showed that PPM1H inhibited the proliferation,migration,invasion and EMT of HCC cells.The in vivo effect of PPM1H on HCC was investigated by tumor formation in nude mice and induced HCC mouse models,and PPM1H inhibited the progression of xenograft tumor and induced liver cancer.Then Co-IP,Western Blot and in vitro dephosphorylation assays were performed to verify that PPM1H directly interact with RPS6KB1 and down-regulates the phosphorylation level of p-RPS6KB1.HepG2 and Huh7 cells were treated with RPS6KB1 inhibitor PF-4708671 and Transwell assays were performed.It was found that PF-4708671 blocked the inhibitory effect of PPM1H on the migration and invasion of HCC cells.Immunohistochemistry was performed to detect the expression of PPM1H in liver tissues of HCC patients.The results showed that the expression level of PPM1H in tumor tissues was lower than that in adjacent tissues.Cross-analysis of the PPM1H expression level and clinical characteristics of patients showed that low expression level of PPM1H was associated with poor prognosis of HCC.Approximately 50%cases of HCC are associated with chronic hepatitis B virus(HBV)infection,which induces acute or chronic hepatitis B(CHB).Although the universal vaccination of HBV vaccine has greatly reduced the probability of HBV infection or development into chronic disease,about 296 million people in the world are still suffering from CHB.At present,the first-line medicines for CHB treatment include interferon(IFN)and HBV reverse transcriptase inhibitors.However,the integration of HBV genomic DNA and cccDNA into the host genome makes it difficult to completely eliminate HBV,and most patients need long-term medication,which tends to cause side effects or drug resistance.ADAR1 can be induced by IFNa and play a promoting role in the HBV infection.Previous studies have found that ADAR1 down-regulates the expression of TTPA and MAVS through RNA editing.MAVS was proved to regulate the innate immune response and promote the activation of interferon regulatory factors IRF3 and IRF7.While TTPA play an anti-HBV role by activating mTORC1 pathway.In order to verify the anti-HBV infection effects of TTPA and MAVS downregulated by ADAR1,and to verify the feasibility and effect of TTPA as an anti-HBV therapy,so as to provide a potential new treatment strategy for HBV infection,the ADAR1 inhibitor 8-azaadenosine(8-aza)was added into the culture medium of HepG2.2.15 cells.The levels of HBV biomarkers were decreased validated by ELISA and qRT-PCR.Western Blot showed that the expression level of TTPA protein was upregulated.TTPA protein was purified and added into cell culture medium,Western Blot assays confirmed that TTPA purified protein could enter the cytoplasm and mouse liver tissues.HBV infected NTCP-HepG2 cells were treated with TTPA protein,ELISA and qRT-PCR showed that the HBV biomarkers decreased after TTPA treatment.TTPA protein was injected intravenously into HBV transgenic(HBV-tg)mice and the elevation of HBV biomarkers in serum and liver tissue of TTPA-treated HBV-tg mice were inhibited.Western Blot assays showed that IFN-α treatment and ADAR1 overexpression inhibited the expression level of MAVS in various HCC cells and mouse primary hepatocytes.MAVS overexpression increased the phosphorylation levels of IRF3 and IRF7.In conclusion,we found that ATF6 inhibited the mRNA and protein expression of PPM1H,and for the first time identified RPS6KB1 as a direct dephosphorylation substrate of PPM1H,which regulates the progression of HCC through dephosphorylating RPS6KB1.The expression level of PPM1H might be a potential molecular biomarker to predict the prognosis of HCC patients.ADAR1 plays a promoting role in HBV replication of HepG2.2.15 cells,and can down-regulate the protein expression level of TTPA and MAVS which have anti-HBV infection activities.TTPA purified protein exhibited anti-HBV infection effect both in vitro and in vivo.Supplementation of TTPA in vivo might be an effective strategy for antiviral therapy of HBV infection.