Study On The Mechanism Of Long Noncoding RNA HOTAIR Regulating The Lipid Accumulation In Nonalcoholic Fatty Liver Disease

BackgroundNon-alcoholic fatty liver disease(NAFLD)is the most common chronic metabolic disease in the world with increasing incidence and is characterized by the accumulation of lipids in the liver.LncRNAs can regulate gene transcription through endogenous competitive regulation(ceRNA)and RNA-binding proteins(RBPs),and have extensive regulatory roles in cell differentiation,development,metabolism,and tumors.The lncRNA HOTAIR has been shown to be involved in cellular metabolism,promote liver fibrosis and lead to accelerated liver carcinogenesis,however,it is unclear whether it also plays a role in the occurrence and progression of NAFLD.Bioinformatics analysis found that HOTAIR had interaction sites with miR-130b-3p,SRSF1 and their downstream target genes related to fat metabolism.Therefore,we propose a hypothesis and explore the role and mechanism of HOTAIR in the occurrence and development of NAFLD,which is expected to find new targets for the prevention and treatment of NAFLD.PurposeThis study intends to build an in vitro and in vivo model of NAFLD to explore whether HOTAIR can target the ROCK1 protein through miR-130b-3p and regulate lipid formation through the two pathways of the SRSF1/MLXIPL axis.Analysis of the role of HOTAIR in lipid formation in NAFLD.Method1.Construct an in vitro and in vivo model of NAFLD,and predict the potential miRNAs and target proteins of HOTAIR through bioinformatics analysis;construct a HepG2 cell model to detect the expression of HOTAIR and the expression changes of downstream genes and proteins after knockdown.The interaction between HOTAIR and miR-130b-3p was analyzed by RIP and dual luciferase reporter system;the HOTAIR,miR-130b-3p and lipid synthesis-related signaling pathways in liver tissue were detected after HOTAIR was silenced in the NAFLD mouse model.express the situation.2.Construct a HepG2 cell model and detect the expression of HOTAIR and SRSF1;bioinformatics analysis predicts the potential targets of HOTAIR,SRSF1 and downstream pathways;RNA-pull down and RIP verify the interaction between HOTAIR and SRSF1,SRSF1 and MLXIPL;knockdown After reducing SRSF1 or HOTAIR,the expression of MLXIPL was detected and the changes of lipid accumulation were observed.3.The expression levels of the above genes and proteins were determined by qPCR and western blot.All experimental data were sorted and analyzed by Excel,SPSS and Image J.Result1.Compared with the control group,the expression levels of HOTAIR and ROCK1 increased in the liver tissue of HFD mice and in the FFA-induced cell model group,while the expression level of miR-130b-3p decreased.Bioinformatics analysis found that HOTAIR has a competitive binding site with miR-130b-3p,and ROCK1 is its potential downstream target.Both RIP and luciferase reporter systems suggested that HOTAIR could play a regulatory role through miR-130b-3p.After knockdown of HOTAIR expression,the above-mentioned changes in the liver and HepG2 cells of the mice caused by high-fat diet or FFA were inhibited;after further inhibition of miR130b-3p in the cell model,the inhibitory effect of knockdown of HOTAIR could be reversed.Overexpression of miR-130b-3p inhibited FFA-induced protein level changes,while further overexpression of ROCK1 reversed the protein level changes caused by overexpression of miR-130b-3p.2.On the other hand,in FFA-induced HepG2 cells,the expression of SRSF1 was increased,the cell viability was decreased,and the lipid deposition was increased.Bioinformatics analysis found that HOTAIR interacts with SRSF1 and MLXIPL,which was verified by RNA-pull down and RIP.In the FFA cell model,knockdown of HOTAIR or SRSF1 resulted in increased MLXIPL gene and protein expression levels,decreased cell viability,and increased lipid accumulation induced by FFA,which were reversed by further overexpression of MLXIPL.Conclusion1.HOTAIR is up-regulated in NAFLD and is associated with lipid formation;2.HOTAIR regulates AMPK pathway through miR-130b-3p/ROCK1 axis to promote lipid formation in nonalcoholic fatty liver cells;3.HOTAIR promotes lipid formation in NAFLD cells by recruiting SRSF1 to stabilize MLXIPL mRNA.