LncRNA LINC00240 Attenuates Fibrogenesis In NAFLD By Inducing Apoptosis Of Hepatic Stellate Cells

Background:In recent decades,about a quarter of the population worldwide suffered from nonalcoholic fatty liver disease（NAFLD）.The latest data showed that NAFLD has become a major public health problem in Asia,with the prevalence rising to 34%.In the future,NAFLD may surpass viral hepatitis as the main cause of liver cirrhosis and hepatocellular carcinoma（HCC）.However,the pathogenesis of NAFLD is poorly understood,and there is still a lack of clear drug treatment strategy.At the same time,unlike other chronic liver diseases,approximately 20-30%of NAFLD-related HCC cases occur in the absence of cirrhosis,which is still poorly studied.Long non-coding RNAs（lnc RNAs）play important roles in the pathophysiology of various diseases.More and more studies have shown that lnc RNAs might be closely related to the progression of NAFLD,but their potential functions in NAFLD remain unclear.The potential mechanisms associated with lnc RNAs have not been fully explored.Therefore,exploring the mechanisms of lnc RNAs in NAFLD can provide us with new perspectives that can help to develop new diagnostic or predictive biomarkers and therapeutic targets to improve its clinical outcomes.Objectives:Novel functional lnc RNAs were identified by constructing a NAFLD-related lnc RNA-mi RNA-m RNA network（NAFLD-related lnc RNA-mi RNA-m RNA network,NLMMN）.Then,human liver tissue and NAFLD cell model were used to verify NAFLD-related lnc RNAs furtherly.Next,the functions and mechanisms of lnc RNAs in NAFLD were explored in vitro.Finally,the fibrogenesis tendency of NAFLD was assessed by constructing a mouse acute fibrosis model.Methods:1.The"Limma R package"was used to identify differentially expressed lnc RNAs（DElnc RNAs）and m RNAs（differentially expressed m RNAs,DEm RNAs）based on the GSE107231 dataset in the GEO database."mi Rcode online tool"and"multi Mi R R package"were used to predict potential interactions between DElnc RNAs or DEm RNAs and mi RNAs,respectively.NLMMN was visualized by Cytoscape.Gene Ontology（GO）,Kyoto Encyclopedia of Genes and Genomes（KEGG）enrichment analysis and protein-protein interaction（PPI）network were performed on DEm RNAs and the m RNAs in NLMMN to explore the potential functions of NAFLD-related lnc RNAs.2.Real-time quantitative reverse transcription polymerase chain reaction（q RT-PCR）was used to identify NAFLD-related functional lnc RNAs in human liver tissue.The NAFLD cell model was constructed by using the hepatocytes cell line Hep G2 and the hepatic stellate cell line LX2,respectively.The expression of NAFLD-related functional lnc RNA was verified by q RT-PCR.3.The effects of the aim lnc RNA on lipid deposition,ROS and apoptosis in hepatocytes and stellate cells were explored by constructing overexpressing and knocking down cell lines of the aim lnc RNA.And the mechanism was further explored using dual luciferase experiments and PCR array.4.The NAFLD model was established in C57BL/6J male mice fed with high-fat diet（HFD）,and C57BL/6J male mice fed with normal diet（ND）served as control group.Then,liver tissue sections of mice were stained with hematoxylin-eosin（HE）and Sirius red to observe the fibrogenesis tendency of NAFLD in mouse acute fibrosis model induced by CCl₄.Results:1.There were 336 DElnc RNAs（154 upregulated and 182downregulated,|log2（fold change）|>0.655 and P?<?0.05）and 399DEm RNAs（152 upregulated and 247 downregulated,|log2（fold change）|>0.608 and P?<?0.05）identified from the GSE107231 dataset.A total of142 DElnc RNA-mi RNA interaction pairs and 643 mi RNA-DEm RNA interaction pairs were involved in the construction of NLMMN,including19 lnc RNAs,47 mi RNAs and 228 m RNAs.The GO analysis results of the m RNAs in NLMMN indicated significant enrichment in five GO terms（collagen-containing extracellular matrix,basement membrane,integrin complex,protein complex involved in cell adhesion,extracellular matrix structural constituent）（P<0.05,Benjamin&Hochberg-corrected）.Meanwhile,only one pathway（ECM-receptor interaction）was found to be enriched（P<0.05）by KEGG enrichment analysis of the DEm RNAs in NLMMN,which was downregulated.It was consistent with the results of KEGG analysis of DEm RNAs.It is suggested that liver fibrosis in NAFLD may be in a suppressed state,and lnc RNA plays an important role in it.2.Two up-regulated lnc RNAs（LINC00240 and RBMS3-AS3）and one down-regulated lnc RNA（ALG9-IT1）were identified by q RT-PCR in human NAFLD liver tissue.And the expression and function of DElnc RNAs were further explored by cell models.LINC00240 was verified in Hep G2 cell line and LX2 cell line.It was worth to notice that LINC00240 was expressed in a low abundance in hepatocytes,but in a high abundance in liver stellate cells.It suggests that LINC00240 might be associated with the development of liver fibrosis in NAFLD.3.It was showed that the ROS content in Hep G2 increased after overexpression of LINC00240,while the ROS content decreased in LX2.The result of flow cytometry showed that overexpression of LINC00240could induce LX2 apoptosis,while knockdown of LINC00240 expression could inhibited LX2 apoptosis significantly.PCR Array results showed that LINC00240 may induce LX2 apoptosis and alleviate liver fibrosis in NAFLD by down-regulating CTSS expression.4.The NAFLD model was established in C57BL/6J male mice fed with high-fat diet（HFD）successfully.The body size and liver volume of HFD-fed mice were higher than those of ND-fed mice.The results of HE staining of liver tissue slides after CCl₄-induced acute liver injury showed that intracellular lipid droplets were significantly increased.The liver tissue slides were stained with Sirius red and analyzed,the results showed that the formation of collagen was significantly higher in the HFD（olive oil）group than that in the ND（olive oil）group（P<0.05）;the HFD（CCl₄）group was significantly higher than ND（CCl₄）group（P<0.01）.ND（CCl4）and HFD（CCl₄）groups was significantly higher than ND（olive oil）（P<0.01）and HFD（olive oil）groups（P<0.001）,respectively.Next,according to the normal reference interval of mice,ALT:25-60 U/L and AST:50-100 U/L,the mice were divided into normal liver function group and abnormal liver function group.There was no significant difference in collagen deposition between HFD（CCl₄）group vs.ND（CCl₄）group group,ND（olive oil）group vs.HFD（olive oil）group,and HFD（CCl₄）group vs.HFD（olive oil）group in normal liver function group.However,the collagen deposition in ND（CCl₄）group was significantly higher than that in ND（olive oil）group（P<0.05）.These results showed that although the collagen deposition in over HFD-fed group was significantly higher than that in ND-fed group,the HFD-fed mice with normal liver function did not show more collagen deposition than ND-fed mice with normal liver function.It was suggested that HFD-fed mice with normal liver function were less responsive to acute liver injury than ND-fed mice with normal liver function.NAFLD with normal liver function is generally considered to be nonalcoholic fatty liver（NAFL）.These results suggest that there is no significant fibrosis tendency in NAFL.Conclusion:1.NAFLD may not have a remarkable tendency of fibrogenesis,it might be mainly regulated by lnc RNAs,and LINC00240 may play an important role.2.LINC00240 could induce stellate cell apoptosis by downregulating CTSS expression,to inhibited the fibrogenesis of NAFLD.3.The fibrogenesis of liver in NAFLD might even be in a state of inhibition,which mostly occurs in NAFL.