| Objective:Non-alcoholic fatty liver disease?NAFLD?is one of the most common chronic liver diseases in the world.The earliest pathological manifestation is hepatic steatosis,which can develop into non-alcoholic steatohepatitis?NASH?.The highest risk of liver fibrosis can progress to cirrhosis and hepatocellular carcinoma.The molecular mechanism of the disease is unclear.In this study,lncRNAs differentially expressing in rabbit steatosis serum and liver tissue were screened by chip technology.lncRNA-uc007gqg.1 was analyzed for gene biological information.By exploring its expression pattern and regulating the endoplasmic reticulum stress and mitochondrial autophagy,it is necessary to find biomarkers and potential intervention targets for NAFLD.Methods:1.High-ThroughputlncRNA chip technology was used to screen and analyze lncRNAs in steatosis serum and liver tissue of rabbit.Gene biology information was analyzed for uc007gqg.1.2.Eighty 8-week-old New Zealand male white rabbits were randomly divided into two groups:the control group?n=40?and the experimental group?n=40?.The blood biochemical indexes of rabbits in each group were measured at 1 week and 6 weeks of the model respectively.The pathological features of liver tissue were collected and the expression levels of lncRNA-uc007gqg.1 and GRP94,Caspase-3 and other proteins in steatosis serum and liver tissue were detected by real-time quantitative pcr and western-blot,respectively.The expression levels of GRP94 and Caspase-3 protein in serum were measured by ELISA.3.SiRNA target screening was succeeding.6 groups were chosen,they are ?SO?cell+sodium oleate?;?SO-Ctrl?cell+sodium oleate+invalid control SiRNA?;?Neg?cell without any treatment?;?Ctrl?cell+invalid control SiRNA?;?Neg-Si?cell+siRNA effective target?;?SO-Si?cell+sodium oleate+siRNA effective target?;TG kit was used to detect triglyceride content;Oil red O staining was used to observe the lipid deposition in each group;The expression levels oflncRNA-uc007gqg.1 and GRP94,Caspase-3 and other proteins were detected by pcr and western-blot,respectively.Results:1.The results of microarray showed that there were 78 lncRNAs with more than 5 times higher expression in fatty liver tissue,and 132 lncRNAs with lower differential expression of more than 5 times.Further analysis revealed that there were 3 IncRNAs?uc007gqg.1,NR028591,uc029syw.1?Not only the difference folds of the three IncRNAs in the respective differential expression profiles are large?differential multiples are?5?,but also they differentially expressed in serum and liver differential expression profiles.Analysis of genetic information revealed that uc007gqg.1 and GRP94 are adjacent to each other in human or in rabbit and mouse genomes.2.Animal experiment results:The blood biochemical index of the experimental group was significantly higher than that of the control group,showing time-dependent and significant difference?P<0.05?.Compared with the control group,the levels of ALT,AST,TC,LDL and HDL in the experimental group began to raise at 1 week,and the increase was the highest at 4 weeks,and the increase was lower at 6 and 8 weeks.The difference was significant?P<0.05?.;The levels of ALB,TB and CRP increased at 1 week,and the highest at 6 weeks.At 8 weeks,the degree of elevation was low,showing a dynamic change,and the difference was significant?P<0.05?.The ratio of GGT,TG,ALT and AST began to increase at 1 week,and was lower at 4 and 6 weeks.The highest was at 8 weeks,and the difference was significant?P<0.05?.The lipid droplet deposition in hepatic cell increased at the 6th week,the fat-like change increased significantly,and the balloon-like degeneration was more significant?P<0.05?.Compared with the control group,the expression levels of uc007gqg.1 gene,GRP94 and Caspase-3 protein in the serum and liver tissues in the experimental group increased significantly and showed dynamic changes,which increased at the 4th week firstly,and increased more significantly at the 8 week compared with the 4 week?P<0.05?,uc007gqg.1 and GRP94 were positively correlated in serum and liver tissues?serum:r=0.996,P=0.004,P<0.05;liver tissue:r=0.985,P=0.015,P<0.05?,uc007gqg.1 and Caspase-3 were positively correlated in serum and liver tissues?serum:r=0.968,P=0.007,P<0.01;liver tissue:r=0.977,P=0.004,P<0.01?.Compared with the control group,the relative expressions of PINK1 and Parkin protein in the liver tissue of the experimental group showed a decreasing trend at each time point,which showed a dynamic change.The expression of these two proteins decreased most at the 8th week when compared with the 4th week and the 6th week,respectively?P<0.05?.The expression of uc007gqg.1 gene and PINK1 and Parkin protein in liver tissue showed opposite trends,the gene and PINK1 were negatively correlated?r=-0.888,P=0.044,P<0.05?;the gene and Parkin were negatively correlated?r=-0.961,P=0.009,P<0.01?.Compared with the control group,the relative expression of p62 protein in the liver tissue about the experimental group increased at each time point,showing a dynamic change,the most obvious increase at the 8th week?P<0.05?,the expression of uc007gqg.l gene and p62 protein in liver tissue showed the same trend and were positively correlated?r=0.911,P=0.032,P<0.05?.3.SiRNA target screening succeeded.In vitro model of NAFLD:Compared with the group of ?Neg?cell without any treatment?,the intracellular TG content was significantly increased in the group of ?SO?cell+sodium oleate??P<0.05?;the number of intracellular red particles increased significantly,and a large number of fat particles were aggregated;the gene of uc007gqg.1 and the two protein GRP 94 and Caspase-3 were significantly increased.The expression level of protein PINK1 and Parkin was significantly decreased,and the relative expression of p62 protein was significantly increased?P<0.05?.Compared with the group of ?SO,there was no apparent intracellular TG content in the group of ?SO-Si?P>0.05?,and the number of oil red particles decreased significantly,and a small amount of fat particles accumulated.The gene of uc007gqg.1 and the two protein of GRP 94 and Caspase-3 decreased significantly in expression?P<0.05?.The expression of PINK1 and Parkin increased significantly,while the relative expression of p62 decreased significantly?P<0.05?.Comparing the group of 1,3 and 6,the increased-expression of the gene uc007gqg.1 was positively connected with the protein GRP94?r=0.901,P=0.014,P<0.05?;the relative expression of uc007gqg.1 was positively connected with the protein caspase-3?r=0.977,P=0.001,P<0.05?.the expression of uc007gqg.l and PINK1 protein were opposite?r=-0.809,P=0.051,P>0.05?;the expression of uc007gqg.1 and Parkin were opposite,they had negative correlation?r=-0.906,P=0.013,P<0.05?;the expression of uc007gqg.1 and p62 showed the same trend,and they were positively correlated?r=0.901,P=0.014,P<0.05?.Conclusion:1.GRP94 may be a target gene for lncRNA-uc007gqg.l to induce hepatic steatosis.2.Rabbit fatty liver model:uc007gqg.1 may be involved in the regulation of GRP94,Caspase-3 and PINK1,Parkin and other proteins in fatty liver cells and is closely related to ERS and mitochondrial autophagy,which may affect hepatocyte apoptosis.It is closely related to the occurrence and development of NAFLD,suggesting that lncRNA-uc007gqg.1 may be a new target for the prevention and treatment of this disease.3.A model in fatty liver cell of NCTC1469 mouse:uc007gqg.1 in vitro hepatocytes may promote hepatocyte lipid deposition in NCTC1469 mice by affecting ERS and mitochondrial autophagy levels to promote apoptosis and injury.It is suggested that lncRNA-uc007gqg.1 may be an early biomarker of NAFLD and a drug target for disease intervention.Both in vitro and in vivo studies suggest that lncRNA-uc007gqg.1 may be involved in the progression of NAFLD disease,but more precise conclusions and mechanisms need to be further expanded in animal and clinical specimens to elucidate the association between uc007gqg.1 and NAFLD disease progression. |
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