Hepatitis B Virus MRNAs Functionally Sequester Let-7a And Enhance Hepatocellular Carcinoma

Hepatocellular carcinoma(HCC)ranks as the fifth most common cancer in incidence worldwide.Hepatitis B virus(HBV)is a major etiological factor in the development of HCC.Viral proteins(e.g.,HBx and HBsAg),chronic inflammation and HBV-DNA integration are thought to be the main inducers of HCC development in Chronic Hepatitis B(CHB).However,the mechanisms remain to be further studied,which may facilitate the development of novel therapy to prevent the development of HCC under chronic HBV infection.We recently proposed a theory of competitive virus RNAs named cvRNAs.HBV mRNAs harbor common miRNA binding sequences with host mRNAs,which enables viral mRNAs to compete with their counterpart host genes for a shared pool of common miRNAs.Given that HBV usually generates highly redundant transcripts upon infection,we expect that direct interactions between viral and host mRNAs may help to elucidate the molecular basis for HCC development and progression.In the present study,we investigated whether highly expressed HBV mRNAs could directly upregulate host mRNA expression via shared miRNA response elements and contribute to the development of HCC.We focused on the prominent tumor suppressor let-7a,a member of the highly conserved let-7 family,as it is the most abundant one among those predicted host miRNAs which simultaneously target genotype C and D HBV transcripts in the liver,and its expression is obviously downregulated in HBV-related HCC.First,by analysis of potential HBV mRNA-regulated miRNAs and host mRNA profiles,we revealed that HBV pre-C/C mRNA leads to upregulation of multiple let-7a targeted genes.Subsequently,a putative let-7a complementary region from nt 86 to 108 in the HBV genome was found in HBV pre-C/C,pre-S,and S mRNAs,and was then verified by site-directed mutagenesis and dual luciferase reporter gene detection system.The let-7a sequestration effect by HBV mRNAs was observed under transfection and virus infection,which is dependent on the let-7a response sequence.Moreover,the copy number of HBV mRNAs is approximately 100-fold more than that of let-7a in HBV-transfected Huh7 cells.In addition,we found reduced AG02 binding,as well as functional mRNA and protein de-repression of let-7a targets(e.g.,c-myc,K-RAS,and CCR7),upon viral mRNA expression.The down-stream signal pathways of let-7 targets were subsequently de-repressed.Let-7a levels in the liver were significantly decreased in HCC patients with HBV infection and were negatively correlated with intrahepatic pre-S2 mRNA levels.Finally,both in vitro and in vivo studies demonstrated that let-7a inhibition by HBV mRNAs resulted in enhanced HCC cell colony formation and tumor growth,providing evidence of the oncogenic potential of HBV mRNAs.Collectively,our results suggest that the host let-7a could be inhibited by viral mRNAs and that up-regulation of its targets plays an important role in facilitating hepatocytes malignant growth in CHB-induced HCC development and progression.Our work thereby demonstrates the reciprocal interplay of host miRNA sequestration and depletion by viral mRNAs.Given the broad interactions of miRNAs and their target mRNAs in viral infections,our data may expand the knowledge of how viral factors modulate host cell signaling pathways in multiple ways,and provide a rationale for the development of novel therapy to prevent the development of HCC under chronic HBV infection.Our results suggest that the interaction between the virus and the host is not limited to protein factors,and that there may be a direct interaction between the virus and the host mRNA.