| Objective:More than 80%cases of Hepatocellular Carcinoma(HCC)in our country are caused by hepatitis B virus(HBV).At present,the carcinogenic mechanism of hepatitis B virus remains unclear.The X protein(Hepatitis B virus X proterin,HBx)encoded by HBV is not only a key transcription factor regulating the transcription and replication of hepatitis B virus,but also the most important effector of HBV carcinogenesis.Studies have confirmed that HBx can promote the proliferation,metastasis,invasion and other malignant behaviorsb of HCC.HBx protein is involved in many epigenetic regulations of host cells to influence the occurrence and development of liver cancer.Recently,our studies have shown that HBx protein can up-regulate and recruit host’s histone methy’ltransferase core subunit(WD repeat domain 5,WDR5)protein to chromatin,promoting oncogene expression of both host cell and HBV,with the epigenetic modification of histone H3 4 lysine trimethylation(H3K4me3).However,WDR5 does not catalyze H3K4me3 directly,which is catalyzed by KMT2s family members.At present,it is not clear which histone methyltransferase is involved in HBx-WDR5-mediated H3K4me3 modification in HBV-induced HCC.In addition,it’s still unknown what are the target genes redulated by "HBx-WDR5-H3K4me3" and how H3K4me3 modification of target genes is up-regulated by HBx needs to be further studied.By re-analysising the studies’ data,we found that HBx and WDR5 bind to the promoter regions of ALKBH5 and FTO genes,and H3K4me3 was enriched in the promoter regions of ALKBH5 and FTO genes.ALKBH5 and FTO are enzymes that catalyse the removal of N6-methyladenosine(m6A)modification from RNA,and they were reported to closely relate to the proliferation of various tumor cells.All these results suggested that the axis of HBx-WDR5-H3K4me3 in HBV-induced hepatocellular carcinoma may affect the modification of RNA m6A enzyme,ALKBH5 and FTO,thus promoting the liver cancinogenesis.m6A modification is widespread and mediates mRNA stability,karyoplasmic transport,RNA splice and translation.Literatures have confirmed that the m6A modification along with its Writers,Erasers and Readers are involved in the occurrence and development of HCC,but there have been not reports the expression of ALKBH5 and FTO in HBV-induced HCC,tumor biological functions and mechanism are still unclear.In addition,it has been reported that the 3 ’untranslated region(3’ UTR)of all HB V RNA contain m6A modifications(including HBx mRNA),which mediate the stability of HBV RNA and HBV’s reverse transcription process.Thus,whether ALKBH5 and(or)FTO,which abnormally expressed in HBV-related tumor cells,can also regulate the RNA methylation modification of HBV itself,remains to be further studied.In all,we tried to explore the role of RNA m6A demethylase ALKBH5 and(or)FTO in HBV carcinogenesis from the epigenetic histone methylation and RNA methylation modification perspectives,clarified how "HBx-WDR5-H3K4me3"up-regulates ALKBH5 and(or)FTO,and finally explored whether ALKBH5 and(or)FTO participate in the regulation of HBx mRNA through the m6A modification.The role of m6A modification in HBV carcinogenesis is supposed to reveal new mechanisms in the development of HCC,and provide new targets to in HCC treatment.Methods:The first part,we investigated the role of mRNA m6A demethylase ALKBH5/FTO in hepatitis B virus-induced liver cancer in China(HBV-HCC).The protein expressions of ALKBH5 and FTO were verified in liver cancer cell lines.The mRNA and protein expressions of ALKBH5 and FTO were verified in 9 clinical samples of hepatitis B induced liver cancer.The immunohistochemical chip of 79 cases of hepatitis B induced liver cancer tissues was used to further verify the expression of the two proteins and the influence of clinical prognosis.Bioinformatics method was used to mine literature data and analyze the expression of ALKBH5/FTO in HBV-induced liver cancer,and their correlation with clinical prognosis.CCK-8,Transwell and nude mice tumor-forming experiments were conducted to evaluate the tumor biological function of HBV-related high expression of ALKBH5/FTO.The second part,we explored which histone methyltransferase in HBV-induced HCC cells is involved in HBx-WDR5-mediated H3K4me3 modification to up-regulate ALKBH5 and FTO.In hepatoma cell model,HBx-WDR5-H3K4me3 axis was verified by RT-qPCR and ChIP-qPCR to up-regulate ALKBH5 and FTO.Through immunoprecipitation and mass spectrometric analysis,we tried to find the histone methyltransferase,and explored the correlation of histone methyltransferase with HBV-HCC.The third part,we verified whether the abnormal expression of ALKBH5 and(or)FTO in tumor cells could also regulate the RNA methylation modification of HBV itself.In liver cancer cell model,the role of ALKBH5 and(or)FTO in regulating the demethylation of HBx mRNA m6A was confirmed by MeRIP-seq and MeRIP-qPCR.Actinomycin D transcriptional inhibition assay was used to further verify the regulation effect of ALKBH5 and(or)FTO on HBx mRNA.Results:1.High expression levels of ALKBH5 and FTO in HCC cell lines and HCC tissues induced by HBVWe found that the levels of ALKBH5 and FTO protein in HBV-infected cells were significantly up-regulated;further,the levels of ALKBH5 and FTO protein were found to be increased in HepG2-NTCP and PHH cells after infected with HBV virus;Among the 9 HBV-positive liver cancer patients,the levels of ALKBH5 and FTO mRNA and protein were significantly higher in tumor tissue than peritumor tissue;IHC further verified that ALKBH5 and FTO proteins were highly expressed in the nucleus of HCC cancer cells;FTO and ALKBH5 mRNA were extracted from 159 patients and protein levels to expand the number of cases,it further clarified that the mRNA and protein levels of ALKBH5 in HCC are significantly higher than those in adjacent tissues,but the protein levels of FTO in cancer and adjacent tissues are not statistically significant;to further confirm that ALKBH5 and FTO are expressed in HBV-related HCC,we conducted histochemical chip assay which showed that ALKBH5 and FTO were highly expressed in 79 cases of HBV-HCC tissues.It is clear that HBV can up-regulate ALKBH5 and FTO in HCC cell lines and HCC tissues.2.The high expression of ALKBH5 and FTO in HBV-HCC affected the clinical prognosisThrough the survival analysis of 159 Chinese cases of HBV-HCC data and 79 cases of histochemical chip results,we found that the higher expression of ALKBH5 and FTO has a poor prognosis of HBV-HCC patients compared to the lower expression group.3.ALKBH5 and FTO promoted the proliferation and metastasis of HCC cellsCCK-8 experiments and tumor migration of transwell experiments confirmed that ALKBH5 and FTO promote the proliferation and metastasis of liver cancer cells.After subcutaneous transplantation of liver cancer cells in nude mice,it was found that both ALKBH5 and FTO can promote tumor formation.4.HBx-WDR5-H3K4me3 axis up-regulated ALKBH5 and FTOOverexpression of HBx increased ALKBH5 and FTO mRNA and protein level while knovkdown of HBx decreased their mRNA and protein level.ChIP-qPCR was used to detect the decreased expression of ALKBH5 and FTO in the gene promoter region H3K4me3 after knocking down either HBx or WDR5.5.WDR82-KMT2F/G participates in the axis of HBx-WDR5-H3K4me3Mass spectrometry detection was conducted after WDR5 protein immunoprecipitation in overexpressed HBx HepG2 cells,the results revealed that WDR82 protein was highly enriched in HBx-WDR5 partner protein;protein immunoprecipitation and western blot experiments confirmed that WDR82 and WDR5 are in the same protein complex;According to the literature reports,WDR82 is the unique subunit of KMT2G/F histone methytransferase complex and KMT2G/F may be involced in the HBx-WDR5-H3K4me3 axis.ChIP-qPCR results indicated that H3K4me3 modification levels of ALKBH5 and FTO gene promoter region were down-regulated in the HepG2.2.15 cells after knocked down WDR82,KMT2G,KMT2F,respectively.Thus,it shows that WDR82-KMT2F/G does participate in the H3K4me3 epigenetic modification of ALKBH5 and FTO.6.WDR82-KMT2G/F was correlated with ALKBH5/FTO expression in HBV-HCC specimensBy analyzing the expression and prognosis of WDR5,WDR82,KMT2G/F from the 159 HBV-HCC Chinese cases,and compareing their correlation with the expression of ALKBH5/FTO,we found that WDR5,WDR82,KMT2G/F may participate in the high expression of ALKBH5 and FTO in HBV-HCC.7.MeRIP-seq confirmed that HBV mediated globle host cell m6A modificationMeRIP-seq and RNA-seq were used to detect m6A modified genes and differentially expressed genes in HepG2 and HepG2.2.15 cells.The expression difference and m6A modification difference genes were intergratively analyzed by Gene Ontology(GO)enrichment analysis and KEGG pathway analysis,which indicated there are globle change of gene expression and m6A modification.8.HBx mRNA has m6A modification,and its m6A modification is down-regulated in HBV-HCC.Mapping the MeRIP-seq data of HepG2.2.15 cells to the HBV genome,we found there were three m6A modification peaks containing GGAC motifs,which suggested that HBV RNA also has m6A modification;MeRIP-qPCR was used to confirm the m6A modification of HBx mRNA;detection of m6A modification of HBx mRNA in clinical liver cancer tissues and peri-tumor tissues showed that the degree of m6A modification of HBx mRNA in tumor tissues was significantly lower than the peri-tumor tissue.9.m6A modification of HBx mRNA mediated the degradation of HBx mRNAWe constructed HBx 3’UTR plasmid with m6A modification site mutation(MT),transfected the plasmids of both HBx 3’UTR WT and MT to cell line,added Actinomycin D(the transcription inhibitor),and then conducted MeRIP-qPCR experiments.The results validated the m6A modification of HBx mRNA and furthermore,the m6A modification of HBx mRNA influenced HBx mRNA’s stbility significantly.10.ALKBH5 regulates the m6A modification of HBx mRNA,and then regulates its expression.We knocked-down ALKBH5 and FTO in HepG2 overexpressing HBx 3’UTR wild-type(WT)cells,and used MeRIP-qPCR to detect the changes of HBx m6A modification.We found that the m6A modification of HBx mRNA was significantly up-regulated after knocked down ALKBH5 but not FTO,which clarified that ALKBH5 instead of FTO is respondible for the m6A modificauion and stability of HBx mRNA.Conclusion:In summary,the main conclusions and significance of this study are as follows:1.ALKBH5 and FTO were highly expressed in HBV-induced HCC,correlated with clinical prognosis,and played a role in promoting tumor cell proliferation and metastasis in HBV-HCC.2.HBx protein encoded by HBV hijacks WDR5-WDR82-KMT2F/G complex of host cells to promote H3K4me3 modification in the gene promoter region of mRNA demethylase ALKBH5 and FTO,and then up-regulate the expression of ALKBH5 and FTO.The up-regulated ALKBH5 and FTO,acting as mRNA m6a demethylases,extensively regulated the m6A modification of host mRNA and mediated the up-regulation or down-regulation expression of a series of genes.In addition,the 3’UTR of HBx mRNA was also modified by m6A and regulated by ALKBH5.The high expression of ALKBH5 inhibited the m6A modification level of HBx mRNA,and then inhibited the degradation of HBx mRNA,so that HBx was maintained in the high expression state,and formed the positive feedback loop of "HBx-ALKBH5-m6A HBx".In this study,the high expression of ALKBH5 and FTO in hepatocellular carcinoma caused by chronic hepatitis B was reported,and it was correlated with clinical prognosis.The molecular mechanism of ALKBH5 and FTO in hepatocellular carcinoma induced by chronic hepatitis B was analyzed,and the effect of ALKBH5 and FTO on the m6A epigenetic modification of HBx mRNA was analyzed.The important role of" HBx-ALKBH5-m6AHBx " positive feedback loop in HBV-induced liver cancer was proposed.This study provides a new molecular mechanism,introduces potential targets,and proposes a hypothesis of epigenetic cross-regulation for the prevention and treatment of hepatocellular carcinoma caused by chronic hepatitis B. |
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