Analysis Of PiRNA Expression Spectra In A Nonalcoholic Fatty Liver Disease Mouse Model Induced By Methionine-and Choline-deficient Diet

Objective: Nonalcoholic fatty liver disease(NAFLD)has become a common health issue worldwide,and piRNA has been shown to be differentially expressed in a variety of diseases.However,the potential relationship between piRNA and NAFLD is unknown.In order to provide new and reliable targets and molecular markers for the treatment and condition monitoring of NAFLD,the research was aimed to investigate the potential relationship between piRNA and nonalcoholic fatty liver disease,finally it laid a theoretical foundation for making further study of piRNA in the roles of the pathogenesis of NAFLD.Methods: The NAFLD model of mouse was established by methionineand choline-deficient(MCD)diet and methionine-and choline-sufficient(MCS)diet.After confirming the establishment of NAFLD,the mice were killed.The mice liver tissues were removed to be stained with haematoxylin eosin(HE),and the levels of serum alanine aminotransferase(ALT),aspartate aminotransferase(AST),total cholesterol(TC)and triglyceride(TG)expression were detected.After successful modeling of NAFLD was confirmed,all mice were killed and total RNA in liver tissue was extracted.The integrity of sample RNA was determined by denatured agarose gel electrophoresis,and the purity and concentration of sample RNA were determined by NanoDrop ND-1000.Then the Arraystar MM9 piRNA array was used to conduct subsequent piRNA Gene microarray analysis on qualified liver tissue samples,to study and screen differentially expressed piRNA.And then the Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis were performed on the differentially expressed piRNA screened by microarray.The molecular biological functions and pathways of differentially expressed piRNA that may be involved in the occurrence and development in NAFLD were preliminarily estimated.Then,the P value,raw Intensity and fold change expressed by piRNA in gene chip detection were taken as the basic conditions to screen piRNA with potential research value.The piRNA with potential research value was selected to perform real-time fluorescence quantitative reverse transcription polymerase chain reaction(RT-qPCR)to further verify the expression of piRNA.Results:1.The wet liver weight and body weight of mice in the model group were significantly lower than those in the control group,while the liver index of mice in the model group was slightly higher than that in the control group.2.The pathological results showed that the model of NAFLD was successfully constructed.In the control group,the hepatic lobule structure was orderly and clear,the size of hepatocytes was normal,the cytoplasm was uniform,and there was no lipid droplet deposition in the hepatocytes.However,in the model group,the hepatic lobule and hepatic cord structures were disordered,and the hepatocytes were swollen with unclear boundaries.Lipid droplet deposits of different sizes were observed in hepatocytes and a large number of inflammatory cells could be seen around the lobules and portal veins.3.Compared with the control group,serum AST and ALT in the model group were significantly increased while TG and TC were decreased.4.Compared with the control group,there were 1285 differentially expressed piRNA in the model group,of which 641 piRNA was up-regulated and 644 piRNA was down-regulated.5.We selected 10 piRNAs in the up-regulated group that may have potential research value for GO and KEGG pathway analysis.The result showed their signaling pathways were mainly involved in cancer-related,hippo,Wnt,Cushing syndrome,FoxO,MAPK and so on.6.piRNA DQ566704 and piRNA DQ723301 were selected for RT-qPCR analysis depend on their P value,raw Intensity and fold change.In NAFLD liver tissues,the above two piRNA genes were overexpressed to varying degrees,and the results were basically consistent with the results of gene chip analysis.Conclusion:1.MCD diet can successfully construct mouse NAFLD model.2.The results of piRNA gene chip confirm the existence of piRNA differential expression in NAFLD liver tissue,and the RT-qPCR of piRNA DQ566704 and piRNA DQ723301 further confirm the reliability of the results of the gene chip,indicating that piRNA may be involved in the occurrence and development of NAFLD,but it is still has some limitations,all the conclusions are predicted based on the results from the microarray and bioinformatics assessments.More work is needed to further illuminate the potential mechanisms.