RNA-binding Protein IFIT1 Promotes The Development Of Non-alcoholic Steatohepatitis

Protein-RNA interactions are extensively existed and play important roles in a variety of physiological and pathological processes.Proteins that can bind to RNA and regulate their biological process or function are named RNA-binding proteins.RNA-binding proteins can regulate multiple biological processes of RNA,such as RNA splicing,localization,transport,degradation,and translation.There are two kinds of methods to elucidate RNA-protein complex.One is protein-centered,and the RNA bound to it is identified in cell lysate by immunoprecipitation of RBP and RIP-seq.The other one is RNA-centered.Proteins were purified by RNA pull-down and subsequently identified by mass spectrometry（MS）.Previous studies used oligo（d T）ion exchange chromatography（IEC）to isolate and analyze the proteome to which poly（A）⁺RNA binding,revealing the interactions between some specific RNA-binding proteins and mRNA.Thus,the concept of"RNA-protein interactome"was formed.However,the interaction between mRNA and protein in specific physiological and pathological processes,as well as the screening of RBP that plays a regulatory function,are still in the process of continuous research progress.This study is mainly aimed at the scientific problem of the regulatory mechanism of the onset and development of nonalcoholic steatohepatitis,which has been a concern of our research group for a long time.Through RNA-centered RBP screening method,we explore the mRNA and their binding proteins that have binding changes in the process of NASH and study their functions in NASH and underlying mechanisms,to propose a new mechanism and intervention for NASH.Non-alcoholic fatty liver disease（NAFLD）is progressive diseases mainly characterized by liver fat deposition,starting from simple steatosis.With the deterioration of the disease,it can progress to non-alcoholic steatohepatitis（NASH）,cirrhosis and even hepatocellular carcinoma.In simple steatosis,free fatty acids from adipose tissue lipolysis or intestinal absorption enter hepatocytes,stimulating hepatocyte de novo lipogenesis（DNL）pathway and leading to triglycerides accumulation in cells.In hepatocytes,excessive lipids droplets,in which are neutral lipids such as TGs and cholesterol ester,push nuclei to one side.During this stage there’s no significant hepatocyte injury and no inflammatory cell infiltration.Serum ALT and AST are at normal level.After synthesizing lipid droplets,hepatocytes then form very low-density lipoprotein（VLDL）to secrete lipid droplets into the blood and transport them to adipose tissue and muscles for energy supply.When lipid droplets accumulate excessively in hepatocytes,fatty acids can be per-oxidized in sub organelles such as peroxisomes,resulting in lipo-toxicity,oxidative stress injury and even death of hepatocytes.Then,damaged and dying liver cells recruit circulating inflammatory cells to the liver,i.e.NASH occurs.When the cell damage persists,liver stellate cells can be activated into fibroblasts which synthesize fibers,leading to liver fibrosis and even HCC.The pathological manifestations of NASH are lipid droplets formation in hepatocytes,ballooned hepatocytes transformation,infiltration of inflammatory cells in the liver with varying degrees of fibrosis,and elevation of ALT and AST in serum.Current studies suggest that NALFD is a reversible pathophysiological process,and with the adjustment of lifestyle and diet habits,patients could restore normal liver metabolism.When NASH occurs,the liver damage of patients is in an irreversible state.Therefore,it is of great importance to explore the molecular expression changes during the transition from NAFLD to NASH and the underlying mechanism for the prevention and treatment of NASH.RNA-binding proteins,as important participants in the regulation of mRNA biological processes,play an important role in the occurrence and development of NASH.However,there is no clear research on the panorama of mRNA and protein interaction in the development of NASH,which is the scientific focus of this study at present.That is,to find the RBP whose mRNA binding ability changes during NASH,and further explore its regulatory role and mechanism in NASH,to provide potential theoretical support for the prevention,management,and control of NASH.Through the screening of RBP changing during the development of NASH,we found 170 RBPs that were up regulated.Among those proteins,the binding fold change of IFIT1（interferon-induced protein with tetratricopeptide repeats）ranked second.Our research group has long focused on the relationship between innate immunity and liver diseases,especially interferon and its effects.As a classical interferon stimulating gene,the role of IFIT1 in liver metabolic diseases has not yet been studied,which arouses our interest.When infection occurs,virus-derived nucleic acids and some pathogen-associated molecular patterns（PAMPs）can trigger antiviral immune response.Viral PAMPs are recognized in the cytoplasm or nucleus by specific host pattern recognition receptors（PRRs）,such as toll-like receptors,RIG-I receptors,and DNA sensors.The combination of PAMPs and PRRs in natural immune cells induces the expression of a series of antiviral genes and cytokines,thereby inhibiting viral replication,among which type I interferon plays a key role in antiviral immunity.IFIT family molecules are important ISGs,including IFIT1,IFIT2,IFIT3 and IFIT5 in human cells,and IFIT1,IFIT2 and IFIT3 in mouse cells,which are induced by viral infection or type I interferon to inhibit viral replication and thus produce antiviral effects.IFIT1 locates in the cytoplasm and exerts antiviral effects through three important molecular mechanisms.First,IFIT1 binds to subunits of the translation initiation complex e IF3,preventing the formation and recruitment of 43S.Second,the interaction of IFIT1 with the 40S ribosome subunit eliminated the formation of 48S.Third,IFIT1 directly binds to cap’0 viral RNA without 2-O methylation of the 5’end nucleotide,preventing the recruitment of e IF4E and e IF4F to RNA and inhibiting viral genes expression.We found IFIT1 in the process of NASH by unbiased screening of RNA-protein interactome.Since there has never been a previous study on the regulatory role of IFIT1in NASH,this arouses our interest.To explore the function and mechanism of IFIT1 in the development of NASH,we constructed Ifit1 hepatocyte specific knockout(Ifit1^hep-/-)mice,and used 4-day NASH model induced by choline and methionine deficient combined with high-fat（MCD）diet to explore its function.we found that the vacuoles within the hepatocyte of Ifit1^hep-/-mice were significantly less than those in Ifit1^f/f mice,and the ballooning degeneration in hepatocytes were also significantly less than those in Ifit1^f/f mice.ROS staining showed that the ROS positive area of Ifit1 hepatocyte specific knockout mice was significantly less than that in the control group.TUNEL staining showed that Ifit1knockout could reduce apoptosis of hepatocytes induced by MCD,and the serum ALT and AST levels of Ifit1^hep-/-mice were significantly lower than those of the control group,suggesting that IFIT1 could promote MCD diet-induced NASH.Then,the mouse liver was stained with oil red,and the expression of liver inflammatory factors was detected.The results showed that the lipid deposition in hepatocytes of Ifit1^hep-/-hepatocyte was significantly less than that in the control group,and the expression of liver inflammatory factors were significantly lower in Ifit1^hep-/-mice.Liver Ly6G staining showed that Ifit1^hep-/-mice had slightly more inflammatory cell infiltration in liver,suggesting that IFIT1 may promote the development of NASH by mediating MCD diet-induced lipid deposition in hepatocytes.To confirm this conclusion,we used multiple NASH models,including the MCD1 month model,the choline deficient combined with high fat（CD-HFD）diet for 3months model,and the simple high-fat HFD model.By examining serum markers,liver oil red staining,liver lipid content,and lipotoxic damage in hepatocytes of Ifit1^hep-/-and Ifit1^f/f mice,we found that Ifit1 hepatocyte specific knockout alleviated lipid deposits in primary hepatocytes in all those diet-induced NASH models,thereby alleviating NASH symptoms in mice.Next,the primary hepatocytes of Ifit1^hep-/-and Ifit1^f/f mice and wild-type mice were obtained to test these phenomena in vitro,and the phenotypes of NASH model stimulated by oleic acid（OA）and palmitic acid（PA）alone or in combination,and MCD medium were used.Oil red staining and TG quantitative detection showed that lipid accumulation in Ifit1^hep-/-hepatocytes was significantly lower than that of Ifit1^f/f primary hepatocytes under NASH conditions in vitro,and the cell viability of Ifit1^hep-/-primary hepatocytes was also significantly higher than that of control primary hepatocytes by MTT assay.Then,primary hepatocytes from 8-week-old C57 mice were obtained to verify the NASH model after knockdown Ifit1 in vitro.The results showed that Ifit1 knockdown could also significantly inhibit the deposition of lipid in primary hepatocytes in vitro.After overexpression of IFIT1 in primary hepatocytes,lipid accumulation in primary hepatocytes under NASH condition was significantly higher than that in control group.All these results suggest that IFIT1 can aggravate the development of NASH by promoting lipid accumulation in hepatocytes.To further clarify this phenomenon in human hepatocytes,we constructed IFIT1KO human liver cell line HHL5 and tested above-mentioned outcome in NASH conditions.The results also showed that IFIT1 KO HHL5 cells had less intracellular lipid accumulation under NASH conditions,and the cell viability of IFIT1 KO HHL5was also significantly higher than that of control HHL5 cells by MTT assay.IFIT1 is a classical RNA binding protein.According to previous studies,IFIT1can bind to mRNA without 5’Cap2 2-O methylation modification and inhibit their expression.Cap2 2-O methylation of mRNA requires a special methyltransferase CMTR2（Cap methyltransferases 2）.Through in vivo and in vitro experiments,we confirm that the expression of CMTR2 is reduced in NASH condition.It is suggested that Cap’2 2-O methylation modification of some mRNA is reduced in NASH condition,which may be the potential mechanism of which IFIT1 binds to more mRNA.To further clarify whether IFIT1 promotes NASH in a Cap’2 2-O methylation modification manner,we knockdown Cmtr2 in primary hepatocytes,and the results showed that Cmtr2 knockdown can aggravate lipid accumulation hepatocytes,suggesting that the decrease of mRNA Cap’2 2-O methylation level mediated by the decrease of CMTR2expression plays an important regulatory role in the occurrence and development of NASH.To further clarify which mRNA with reduced Cap’2 2-O methylation were bound by IFIT1 in NASH,RIP-seq of IFIT1 protein and Cap’2 2-O methylation sequencing in NASH were performed.In IFIT1 RIP-seq,molecules which were bound to IFIT1more under OA/PA stimulation and molecules that are less methylated by Cap’2 2-O under OA/PA stimulation are compared and intersected.We got 5 targets,Prdx6（peroxiredoxin 6）,Sqstm1（sequestosome 1）,Nedd8（neural precursor cell expressed,developmentally down-regulated gene 8）,Ambp（alpha-1-microglobulin/bikunin precursor）,Eef1a1（eukaryotic translation）.Then we conducted IFIT1 RIP-q PCR experiment,and the results showed that IFIT1 could directly bind to the mRNA of Prdx6,while Q-PCR and Western blot also proved that the expression of Prdx6decreased under OA/PA stimulation.The above results indicated that,Cap’2 2-O methylation modification of Prdx6 mRNA was reduced in NASH,which enabled IFIT1to bind the mRNA of Prdx6 and inhibit its expression.In summary,through unbiased RNA-protein interactome capture technology,we found that IFIT1 binds to more mRNA during NASH,and IFIT1 hepatocytes conditional knockout could alleviate NASH development in mice under various models by reducing lipid accumulation in hepatocytes.Further mechanism studies showed that,in NASH condition,the expression of CMTR2,a key writer for mRNA Cap’2 2-O methylation modification,decreases the mRNA Cap’2 2-O methylation of Prdx6,a key protein for lipid oxidation,resulting in increased binding of IFIT1 to it,thereby reducing the expression of Prdx6 and blocking lipid oxidation.Excess accumulation of lipid droplets in liver cells ultimately promotes the development of NASH.