A Preliminary Study Of Serum Micro RNAs Expression Differences In The Nonalcoholic Fatty Liver Disease

Part I Screening of Differentially expressed miRNAs in NAFLD patientsObjective:This study intended to screen serum miRNA expression difference spectrum between NAFLD patients and normal healthy people by high throughput sequencing technology,fin order to find out the specific expressed miRNAs in NAFLD patients.Methods:(1)According to the serum collection standard operating procedures,23 NAFLD patients serum and normal serum specimens were collected,making age and gender ratio between two groups as close as possible..All of the patients and volunterrs are informed consent and sign informed consent.(2)Selecting 3 NAFLD patients serum and 3 normal serum specimens,whose age and gender ratio and history of smoking and drinking were similar,as the two filter groups.And the rest serum specimens were for validation.(3)Trizol extraction method was used to extract the total RNA of the serum samples of the two groups,and the purity and concentration of RNA were measured by Nanodrop2000 spectrophotometer.(4)Testing the quality of RNA to construct cDNA library and then Employing the Illumina high-throughput sequencing approach for serum total RNA sequencing in two groups of specimens;(5)By comparing miRBase database miRNAs precursor and mature sequences,to identify and filter the known differentially expressed miRNAs.Results:(1)By Illumina high-throughput sequencing,we filtered 129 differentially expressed miRNA in NAFLD and CTL,which had 103 up-regulation miRNAs,26 down-regulation miRNAs.And 125 of them had been known,while 4 of them were new here.Those unkown miRNAs were hsa-miR-1357-p3,hsa-miR-1237-p5,hsa-miR-1246-3-p5 and hsa-miR-8564-p3.(2)It was found that 4 serum miRNAs in NAFLD patients were 2 fold higher than that in the normal.Hsa-miR-122-5p was 7.26 fold higher in NAFLD patients than the normal.Hsa-miR-1290 was 4.21 fold higher in NAFLD patients.Hsa-miR-192-5p was 3.01 fold higher,while hsa-miR-130a was detceted to be 2.54 fold lower in NAFLD patients than the normal.Conclusion:Significant differently expressed serum miRNAs existed in NAFLD patients.These 4 serum miRNAs could have a close relationship with the development of NAFLD.Part II Identification of serum miRNAsObjective:The expression of miRNA was evaluated by real-time fluorescence quantitative PCR method to evaluate the reliability of miRNA expression profile of primary screening.Methods:(1)Extracting RNA from the serum of NAFLD group and CTL group by RNA extraction kit and testing the purity and concentration of RNA by Nanodrop2000 spectrophotometer(2)Then making a preparation for cDNA by RT reaction.And finally,vrifying the expression levels by qRT-PCR.Results:Compared to the normal,hsa-miR-122-5p was 8.25 fold higher in NAFLD patients,hsa-miR-1290 was 3.14 fold higher,hsa-miR-192-5p was 3.58 fold higher,while hsa-miR-130a was 2.13 fold lower.Conclusion:(hsa-miR-122-5p,hsa-miR-1290,hsa-miR-192-,hsa-miR-130a)These 4 miRNAs expression levels were matched with high-throughput sequencing results,indicating that there were specific serum miRNAs expressed in NAFLD patients.And these miRNAs possibly be related with the development of NAFLD.Part ? Target genes prediction and bioinformatics analysis of serum miRNAsObjective:To explore the mechanism of the effect of serum miRNAs expression on NAFLD,the the target genes of differentially expressed miRNAs were predicted in this part.Methods:TargetScan,Pitar and Miranda these three softwares were used to predict the target genes of differentially expressed miRNA and the role of the target genes were evaluated by GO annotation analysis and KEGG pathway analysis.Results:The function of target genes showed mainly in the molecular functions,components,and biological processes by GO annotation.And these target genes might be related with several pathways,mainly connected to pathways in cancer,MAPK signaling pathway,regulation of actin cytoskeleton,focal adhesion,insulin signaling pathway,wnt signaling pathway,et al.Conclusion:Preliminary predicting the serum miRNAs target genes.These miRNAs might have effect on NAFLD through those predicted taget genes.And the molecular mechanism might be by the insulin signaling pathway,MAPK signal pathway,and wnt signaling pathways or other pathways.