Identification Of MiRNAs Targeting Paraoxonase 1 And Its Clinical Application In Nonalcoholic Fatty Liver Disease

Background:Nonalcoholic fatty liver disease?NAFLD?excludes alcohol consumption and other well-defined liver damage factors.The clinicopathological features are mainly characterized by excessive accumulation of fat in liver cells.Its pathogenesis is still unclear,mainly believed to be related to oxidative stress,insulin resistance and genetic susceptibility,often accompanied by obesity,diabetes,hyperlipidemia,cardiovascular disease and other metabolic related syndromes.As one of the members of the paraoxonase family,the antioxidant enzyme activity of PON1 in lipid metabolism cannot be ignored.It is closely related to the occurrence and development of various diseases.Numerous studies have shown that PON1 plays an important role in the oxidative stress response of the liver.Compared with healthy people,serum PON1activities in patients with NAFLD,liver fibrosis and cirrhosis are all significantly decreased.However,few studies are focused on the changes in gene and protein expression of PON1 in NAFLD,and the reasons for these changes still remain unknown.Objective:?1?Bioinformatics methods were used to screen for possible microRNAs?miRNAs?that targeting PON1;?2?To confirm the regulation effects of miRNAs on PON1 in vitro;?3?To explore the changes of serum PON1 and miRNA levels in healthy people and patients with nonalcoholic steatohepatitis?NASH?as it can provide new ideas and methods for the diagnosis and treatment of NASH patients.Methods:?1?TargetScan7.0 software was used to predict miRNAs that may be associated with PON1;Subsequently,the dual luciferase reporter gene vector was constructed,and the recombinant plasmid was transfected into HEK-293T cells.The fluorescence was hRluc and the corrected fluorescence was hLuc.The ratio of hLuc fluorescence value to hRluc fluorescence value was applicable for representing the relative activity of luciferase detected by the plasmid after transfection and the relevant miRNAs targeting PON1 were initially determined.?2?To up/down-regulated the miRNA levels in HepG2 cells.The specific transfection experiments were as follows:normal cell control group,mimic and inhibitor negative control group,miR140-5p mimic group and its inhibitor group as well as the fluorescence-independent control group.The expression of miRNA in each group was detected by quantitative real-time polymerase chain reaction?qRT-PCR?,and PON1 protein in HepG2 cells was detected by Western blot?WB?to verify miRNA targeting PON1.?3?The serum samples of 34patients with NASH were collected,and 31 healthy subjects were used as the normal control group.The qRT-PCR technique was applied to detect the changes of the miRNAs in the previous two groups,and the enzyme-linked immunosorbent assay?ELISA?was used to detect the protein expression of PON1 in the two groups.Results:?1?Three PON1-related miRNAs were predicted using TargetScan7.0software:miR140-5p,miR183-5p and miR203a-3p.Analysis of the dual luciferase activity suggested that the hsa-miR140-5p,hsa-miR183-5p and hsa-miR203a-3p mimics had significant down-regulation effects on the reported fluorescence of the PON1wild-type vector(P_miR140-5p<0.001,P_miR183-5p<0.01,P_miR203a-3p<0.01).?2?qRT-PCR results of in vitro experiments showed that miR140-5p had higher expression levels in the mimic group compared with the mimic negative control group?P<0.001?while the expression of miR140-5p was not statistically significant in the normal cell control group compared with the mimic negative control group?P>0.05?.When it compared to the inhibitor-negative control group,the expression of miR140-5p was significantly decreased in the inhibitor group?P<0.05?,but the expression of miR140-5p was not significantly different between the normal cell control group and the inhibitor-negative control group?P>0.05?.The WB results suggested that the expression of PON1 protein was significantly down-regulated in the miR140-5p mimic group compared with the mimic-negative control group?P<0.001?,and the expression difference was not statistically significant in the normal cell control group?P>0.05?;Compared with the inhibitor-negative control group,the expression of PON1 protein was significantly up-regulated in the miR140-5p inhibitor group?P<0.01?,and there was no statistically significant difference in the normal cell control group,either?P>0.05?.?3?Studies in patients with clinical NASH indicated that the expression level of miR140-5p was significantly lower in the serum of NASH patients than that of healthy controls?P<0.01?while the opposite result was found in the expression level of PON1?P<0.05?.The area under the ROC curve of miR140-5p was 0.719?95%CI:0.593⁰.845?.When the cut-off value was 0.434,its specificity as a screening indicator for NASH was 85.3%,and the sensitivity was 58.12%.Conclusions:The above experimental results suggest that miR140-5p,as an upstream regulator of PON1,can inhibit the expression of PON1.In the clinical study of NASH patients,the expression level of miR140-5p was decreased,demonstrating that miR140-5p may participate in the progression of NASH through regulation of post-transcriptional gene expression of PON1.