The Role And Mechanism Of Long Non-coding RNAs In Non-alcoholic Fatty Liver Disease

Objective:With the prevalence of diseases such as obesity and metabolic syndromes,the global incidence of non-alcoholic fatty liver disease is also increasing year by year,which brings a great burden to the world.To establish the cell model of nonalcoholic fatty liver disease using LO2 cell induced by oleic acid.Then we explored the differential expression profiles of long non-coding RNAs and mRNAs in cell model through sequencing analysis.Then we screened out a special lncRNA,to further investigate its possible role in non-alcoholic fatty liver disease.Our study provided theoretical support for the clinical value research of IncRNA in NAFLD.Methods:1.The Establishment of non-alcoholic fatty liver disease cell model:Human normal liver cell line LO2 cell was treated with different concentrations of oleic acid(10?g/mL,20 ?g/mL,30 ?g/mL,40?g/mL,50 ?g/mL,60 ?g/mL)and DMSO(normal control groups).After 48h,the inverted microscope and semi-quantitative analysis after oil red O staining were performed to observe the formation of intracellular lipid droplet,determining whether the cell model was successfully established.2.The differential expression profiles of mRNAs and IncRNAs in a non-alcoholic fatty liver disease cell model was obtained through Eukaryotic circular sequencing.3.According to fold difference,IncRNA type,and other criterias,six lncRNAs of interest were selected to further fluorescent quantitative PCR verification,namely IncRNA ENST00000414790,IncRNA ENST00000431095,IncRNA ENST00000608018,lncRNA ENST00000442037,IncRNA ENST00000611525 ?lncRNA ENST00000255183.4.According to Gain-of-function(GOF),LncRNAs whose fluorescence quantitative PCR results were consistent with sequencing(including lncRNA ENST00000255183,ENST00000442037,and ENST00000611525)were selected to construct lncRNAs overexpression plasmids using pcDNA3.1 as the vector,then transfect human normal liver cell line LO2.The fluorescence microscopy and fluorescent quantitative PCR were used to confirm the overexpression of lncRNAs.Then oleic acid(DMSO as control)was used to induce cell model.Oil red O staining and semi-quantitative analysis were used to investigate whether the Gain-of-function of target lncRNAs play a role in the lipid accumulation process of LO25.The ceRNA bioinformatics analysis of lncRNA ENST00000255183 was performed to predict the downstream mRNAs.Futhermore,we intersected the mRNAs predicted by ceRNA bioinformatics analysis and the mRNA expression profile in the results of sequencing.Then qRT-PCR was used to verify.Results:1.Inverted microscope showed lipid droplets with different sizes can be seen in OA groups,with a ring-like distribution from the periphery of the cells.After oil red O staining,the lipid droplets were red.Compared with the control group,the cell viability of OA groups were not significantly statistically significant at the concentrations from 10 ?g/mL to 50 ?g/mL(P>0.05).However the cell viability decreased significantly at 60 ?g/mL,and the difference was statistically significant(P<0.05).The semi-quantitative analysis showed that with the increasing of OA concentrations,the degree of lipid droplet formation in L02 cells also gradually increased,but there was no significant difference after 50 ?g/mL.2.The circular sequencing results of eukaryotes showed that compared with the control group,there were a total of 648 lncRNAs with differential expression greater than 1-fold,of which 351 were up-regulated and 297 were down-regulated.In total,there were 3859 mRNAs with differential expression greater than 1-fold,of which 1884 were up-regulated and 1975 were down-regulated.3.Fluorescence quantitative PCR verified that compared with the control group,the expressions of lncRNA ENST00000414790,lncRNA ENST00000431095 and lncRNA ENST00000608018 showed no significant difference(P>0.05),and the multiples of change were(0.93±0.07),(0.65±0.27),and(0.89±0.20).And the expressions of lncRNA ENST00000255183,lncRNA ENST00000442037,and lncRNA ENST00000611525 in OA group were down-regulated by(0.35±0.07),(0.52±0.13),and(0.38±0.15)folds,the differences were statistically significant(P<0.05),which were consistent with the results of sequencing.4.Effect of over-expression target lncRNAs on NAFLD cell model:qRT-PCR identified the over-expression effect of high-expressing cell lines.Compared with the unloaded plasmid pcDNA3.1,The corresponding lncRNAs expression levels of the lncRNA ENST00000255183+,ENST00000442037+,and ENST00000611525+groups were respectively increased by(4151.36±1316.90),(8946.66±2405.24)and(1605.99±329.68),and the differences were statistically significant(P<0.01).Subsequently oleic acid was used to induct cell model.Then oil red O staining and semi-quantitative analysis experiments found that compared with the control unloaded plasmid group,the overexpression of lncRNA ENST00000442037 and ENST00000611525 did not significantly affect lipid droplet formation(P>0.05);the overexpression of lncRNA ENST00000255183 group had significantly reduced lipid droplet formation,and the difference was statistically significant(P<0.05).5.The ceRNA Bioinformatics analysis of lncRNA ENST00000255183 predicted that 241 mRNAs formed a ceRNA network with IncRNA ENST00000255183.Further GO and KEGG pathway analysis revealed that the adipocytokine signaling pathway was significantly enriched,and the differential gene involved was RXRB.Combined with the sequencing results of the mRNAs expression profile,we intersected with the predicted mRNA.After intersecting,5 mRNAs were obtained,namely RXRB(Retinoic acid receptor RXR-beta),RNPEPL1(arginyl aminopeptidase-like 1),CD82,MADD(MAP kinase-activating death domain protein)and KLC2(Kinesin light chain 2).6.The qRT-PCR results of 5 mRNAs showed that compared with the control group,RXRB,RNPEPL1 and CD82 were down-regulated by(2.24±0.35),(2.55±1.03),and(3.91±0.92)fold,and MADD and KLC2 were up-regulated by(3.75±1.36)and(1.20±0.19)fold in the OA group,which were consistent with the sequencing results.Conclusion:1.50 ?g/mL oleic acid successfully induced NAFLD cell model,which can be used in subsequent experiments.2.The oleic acid-induced NAFLD cell model has extensive differential expression profiles of lncRNAs.3.LncRNA ENST00000255183 participates in the lipid accumulation process of NAFLD,and the mechanism may be as ceRNA to regulate the expressions of RXRB,RNPEPL1 and CD82,which related to adipocytokine signaling pathways.