Investigation Of The Role And Mechanism Of TIP47in NAFLD And Hepatic Steatosis Caused By HCV Infection

Background:Fatty liver disease (FLD) is reversible and mainly has vesicular aggregationof triglycerides in the liver cells, namely steatosis. The incidence rate of FLD indifferent countries is10%~24%. In China, with the change of people’s diet andlifestyle habits due to national economy improvement, the incidence rate of FLDhas significantly increased. Two most common factors for FLD are non-alcoholicfatty liver disease (NAFLD) and HCV infection. FLD caused by NAFLD andHCV infection showed lipid accumulation and lipid droplets (LD) formation inliver cells.The basic structure of LDs is lipid core consisted of the neutral lipids,monolayer phospholipid layer coating the lipid core and the LD surface proteinembedded in the phospholipid layer. At present, the lipid droplets formation is notclear. The most popular hypothesis is that synthesized lipids accumulate in thedouble-layer membrane of the endoplasmic reticulum and increase the volume ofthe membrane structure bulging and forming a globular structure. Finally, the globular structure detaches from the membrane structure and forms independentLDs. In the formation of LDs, some proteins play important roles. LD surfaceproteins PAT family may play a key role.In mammals, the PAT family includes five members: perilipin, ADRP, TIP47,S3-12and OXPAT. PAT family members differ in the molecular size, tissueexpression, LDs affinity, protein stability, and transcriptional regulation. But theyare highly homologous with the N-terminal PAT domain and play a role in theregulation of cell lipid metabolism and LDs formation.In1999, TIP47was first discovered to mediate transport from the endosometo the trans-Golgi network by binding the mannose receptor6-phosphoric acid(MPR). In2001, TIP47was found to be highly similar to the LD protein ADRPand perilipin, function the initial lipid droplet formation, should be aLD-associated protein. But the specific role and molecular mechanism of TIP47in lipid droplet formation and metabolism are not clear.Objective:The purpose of this study was to determine the role of TIP47protein inhepatic steatosis caused by NAFLD and HCV infection, investigste the role ofTIP47protein in cellular LDs formation and lipid distribution in liver cells byinhibiting the expression of TIP47protein, and clarify the role of interaction ofmicrotubule and TIP47. Finally, we hope this study may contribute to understandthe mechanism of steatosis and molecular basis of LDs biogenesis.Methods:1. Rabbit anti-mouse TIP47polyclonal antibody was generated to determinethe expression of TIP47of various tissues (especially the liver) in NAFLD animalmodel of ob/ob mouse by IHC and western blot assays.2. Cell lines with stable expression of HCV core antigen of genotype3a and1b in Huh-7cells were build. MicroRNA expression profile between these twocell lines was determined by microarray and qPCR assays and their target geneswere predicted by computational analysis. Finally, the role of LD-associatedprotein PAT family in liver steatosis induced by HCV core antigen and their expession were performed.3. By inhibiting TIP47expression in AML12cells via siRNA technology,morphological changes of LDs and cellular lipid distribution were observed todetermine the cell biological functions of TIP47. Furthermore, microtubule wasrecruited in the process of LDs biogenesis promoted by TIP47through indirectimmunofluoresence staining.Results:1. Rabbit anti-mouse TIP47polyclonal antibody was successfully preparedand TIP47was associated with NAFLD-related steaosis.The soluble protein consisting of TIP47protein C-terminal398amino acidswas cloned and expressed in E.coli BL21cells. Rabbit anti-mouse TIP47polyclonal antibody was successfully prepared by immunizing New Zealandrabbits using TIP47(117-437aa) protein. In NAFLD animal models of ob/obmouse, we found that the TIP47expression was significantly higher thanwild-type mice in the tissues with active fat metabolism, such as the liver, skeletalmuscle, cardiac muscle and brown adipose tissue. These results indicate thatTIP47plays a role in cellular LDs biogenesis.2. HCV core antigen of genotype3a and1b could induce hepatic steatosis byregulating the microRNA and its target protein. And the PAT family memberTIP47plays a role in HCV-induced steatosis.By microarray and quantitative PCR assays, microRNA expression profileand the differentially expressed microRNAs between Huh-7cells transfected byHCV core antigen of genotype3a and1b were determined. MicroRNA targetgenes and their functions and pathways were analysed, which forecasted the keygenes for hepatic steatosis caused by HCV core antigen3a and1b. Some of themwere fat metabolism and transport associated. By the analysis of PAT familymembers expression in Huh-7cells transfected by HCV core antigen3a and1b,we found the expression of TIP47was increased and ADRP was decreased,which suggested that TIP47play roles in hepatic steatosis and LDs biogenesisinduced by HCV core antigen. 3. TIP47mediated cellular TG transport from the endoplasmic reticulum toLDs and promoted LDs biogenesis by interacting with microtubules.By inhibiting TIP47expression in AML12cells, intracellular LDs decreasedin number and size, TG stored in LDs reduced, intracellular total TG did notchange significantly, the endoplasmic reticulum lumen expanded and TGaccumulated in the ER increased. During inducing LDs using oleic acid inAML12cells, TIP47gradually gathered from the endoplasmic reticulum to LDssurface. In summary, TIP47could mediate TG transport from the ER to LDs.Using indirect immunofluorescence assay, we found that TIP47were co-localizedwith microtubule α-tubulin, which suggested TG transport mediated by TIP47was associated with microtubules.Conclusion:In this study, we succeeded in the preparation of sensitive and specific rabbitanti-mouse TIP47polyclonal antibody. Using this antibody, we found TIP47wassignificantly higher in NAFLD and HCV core antigen-induced hepatic steatosis.We generated the microRNA expression profiles of Huh-7cells transfected byHCV core3a and1b. The target genes of these differently expressed microRNAsbetween core3a and1b were forecasted and some crucial proteins associatedwith lipid metabolism and transport, including TIP47, were determined. Wefirstly identified that TIP47could transport triglycerides from the endoplasmicreticulum to lipid droplets and then promote lipid droplets biogenesis byinteracting with microtubules.