The Role Of MiR-199a-3p And RIG-I In Hepatocarcinogenesis And The Underlying Mechanisms

Primary liver cancer is a kind of common cancer around the world and hepatocellular carcinoma is the most common type of primary liver cancer,which accounts for nearly 90%.There are multiple factors and procedures participating in the hepatocarcinogenesis.Up to now,researches suggest that hepatocarcinogenesis is tightly linked to liver diseases,such as chronic virus hepatitis(HBV and HCV),alcoholic hepatitis or fibrosis,and non-alcoholic fatty liver diseases.In general,long-term chronic hepatic injury,injury-induced chronic liver inflammation,and compensatory hepatocyte proliferation play critical roles in hepatocarcinogenesis.There are plenty of molecules and pathways involved in hepatocarcinogenesis,which make the underlying mechanisms extremely complex.The molecular regulation mechanism is the key scientific question in this field.Part 1.The role of micro RNA-199a-3p in hepatocarcinogenesis and the underlying mechanismHepatocacinogenesis is closely related to hepatic injury.The damage associated molecular patterns(DAMPs)released by the injured hepatocytes not only activate the immune cells in liver,but also recuit the inflammatory cells to the liver,causing liver inflammation.On one hand,inflammation stimulates the compensatory hepatocyte proliferation.On the other hand,it also aggravates the liver damage.This vicious circle makes a chronic inflammatory enviroment in liver.The consistent death of hepatocytes leads to chronic proliferation of hepatocytes,which eventually induces malignant transformation of hepatocytes.As hepatic injury is the first step of this vicious circle,the damage and death of hepatocytes are of great significance in hepatocarcinogenesis.Apoptosis of hepatocytes plays a critical role in hepatocarcinogenesis,several researchers have reported its role in promoting hepatocarcinogenesis.The roles of mi RNAs in tumor progression are intensively investigated,while their roles are relatively less explored in the field of hepatocarcinogenesis.In our previous work,we found that mi R-199a/b-3p was abundantly expressed in human liver tissue,while its expression was markedly reduced in HCC tissue.Further research proved that mi R-199a/b-3p suppressed tumor progession.However,the role of mi R-199a/b-3p in hepatocarcinogenesis remains unknown.Therefore,we constructed the mi R-199a/b-3p deficient mice and investigated its role in hepatocarcinogenesis and the underlying mechanism.In human liver,we previously found that mi R-199a/b-3p was expressed from mi R-199a-2 gene.So,we first investigated the mi R-199a gene in mice.By using CRISPR/Cas9,we constructed the mi R-199a-1 and mi R-199a-2 conventional knockout mice.The results showed that mi R-199a-3p was the mature mi RNA,instead of mi R-199a-5p.Meanwhile,mi R-199a-3p was mostly from mi R-199a-2 gene.Hence,we next constructed the hepatocyte-specific mi R-199a-2 knockout mice.Through the DEN-induced and DEN plus CCl4-induced HCC model,mi R-199a-3p deficiency was found to promote hepatocarcinogenesis.In DEN or APAP-induced liver injury model,mi R-199a-3p was found to enhance the hepatic injury and hepatocyte apoptosis,which was also determined in mi R-199a-2 knockout hepatocytes.Mechanistically,we first explored the way how mi R-199a-3p deficiency promote hepatocyte apoptosis.The results showed that knockout of mi R-199a-3p downregulated the level of anti-apoptotic protein Bcl-XL which made it clear that mi R-199a-3p regulated hepatocyte apoptosis through mitochondrial.Then,proteomic TMT mass spectrometry was conducted in the liver of mi R-199a-2^f/f and mi R-199a-2^hep-/-mice.After analyzing and screening,the expression of PDCD4,a pro-apoptotic protein,was found increased in the liver of mi R-199a-2^hep-/-mice.The m RNA of PDCD4 was not changed but the protein of PDCD4 was increased in mi R-199a-3p deficient mice and cells.Also,the pro-apoptotic role of PDCD4 was comfirmed.Moreover,the results in vitro and in vivo showed that mi R-199a-3p deficiency promoted hepatic injury and hepatocyte apoptosis dependent on PDCD4.Generally,in this study,we showed that mi R-199a-3p inhibited the expression of PDCD4.As PDCD4 was a pro-apoptotic protein,mi R-199a-3p deficiency increased the hepatic injury and hepatocyte apoptosis,which could promote hepatocarcinogenesis.Part 2.The role of RIG-I in hepatocarcinogenesis and the underlying mechanisamHepatic injury and injury-induced inflammation play vital roles in hepatocarcinogenesis.The consistent inflammation stimulates chronic hepatocyte proliferation,causing hepatocytes transformation to malignant cells.During hepatocarcinogenesis,hepatocytes are transformed to Hc PCs and then to established HCC cells under inflammatory circumstances.The molecular regulatory mechanisms in this process are of great interest.The DAMPs released by injuried hepatocytes stimulate the Kupffer cells in liver to secrete proinflammatory cytokine IL-6.IL-6 and IL-6 effector signaling play critical roles in hepatocarcinogenesis,especially in the procedure from Hc PCs to HCC.The autocrine IL-6 from Hc PC promotes its transformation to established HCC.However,the regulation of IL-6 signaling and its function in Hc PCs have not been reported.Previously,we found that RIG-I was expressed in hepatocytes in human liver and its expression were markedly reduced in HCC tissues.Besides,RIG-I improved the effect of IFN therapy by enhancing IFN effector signaling.Meanwhile,the expression of RIG-I in male was much lower than that in female,which was coincident with the high morbidity of HCC in men.However,the role of RIG-I in hepatocarcinogenesis,especially in the transformation from Hc PCs to HCC,still remains unclear,which excites our interest to investigate.We first examined RIG-I expression during hepatocarcinogenesis,including normal hepatocytes,nonaggregated hepatocytes and aggregates containing Hc PCs from mice five months post DEN injection,and established HCC cells.Both protein and m RNA levels of RIG-I was determined to be decreased in Hc PCs and established HCC cells,while its expression between normal hepatocytes and nonaggregated hepatocytes were similar.As IL-6 plays critical roles in hepatocarcinogenesis,especially in Hc PCs,we predicted that the decreased expression of RIG-I may be mediated by IL-6.IL-6 stimulation increased the expression of RIG-I in the short term,while decreased the expression of RIG-I in the long term.Mechanistically,short-term IL-6 stimulation activated STAT3,and then STAT3translocated to nuclear and bound to RIG-I promoter,which promoted RIG-I transcription.Long-term IL-6 stimulation modulated histones methylation,which led to the reduction of RIG-I.We next constructed the hepatocyte-specific RIG-I knockout mice and macrophage-specific RIG-I knockout mice.The results showed that hepatocyte-specific RIG-I deficiency promoted hepatocarcinogenesis in DEN-induced and DEN plus CCl4-induced HCC model,while macrophage-specific RIG-I deficiency did not influence the hepatocarcinogenesis.Then,in DEN-induced hepatic injury model,we found that hepatocyte-specific RIG-I deficiency did not influence the hepatic injury,the expression of inflammatory cytokines,inflammatory cells infiltration,and compensatory hepatocyte proliferation.However,the protein phosphorylation microarray also showed increased STAT3 phosphorylation in RIG-I^hep-/-mice after DEN administration.In RIG-I^hep-/-IL-6^-/-and RIG-I^hep-/-IL-6ra^hep-/-mice,DEN-induced hepatocarcinognenesis was similar with RIG-I^hep-/-mice,which indicated that RIG-I suppressed hepatocarcinogenesis depending on IL-6 signaling.Further study showed that the K18 and K146 demythylation of RIG-I induced by IL-6 stimulation had an important influence on the phosphorylation of STAT3.IL-6 stimulation enhanced the interaction of RIG-I and JMJD4 which was a demethylase and mediated the demethylation of RIG-I,to enhance the suppressive effect of RIG-I on STAT3 phosphorylation.In this study,we found that the expression of RIG-I was down-regulated in Hc PCs and hepatocyte-specific RIG-I deficiency promoted hepatocarcinogenesis.Mechanistically,IL-6 stimulation induced the interation of JMJD4 and RIG-I and then mediated the demethylation of RIG-I in K18 and K146 which promoted the interaction of RIG-I-STAT3and enhanced the inhibitory effect of RIG-I.Yet,hepatocyte-specific RIG-I deficiency completely removed this effect and enhanced the IL-6/STAT3 signaling pathway both in hepatocytes and Hc PCs,which eventually promoted hepatocarcinogenesis.