布鲁氏菌介导的mmu-miR-671-5p的靶基因预测与功能富集性分析

Target Gene Prediction and Functional Enrichment Analysis of mmu-miR-671-5p Mediated by Brucella

为进行布鲁氏菌介导的mmu-miR-671-5p的靶基因预测,本试验利用布鲁氏菌(Brucella)感染RAW264.7细胞后,分别运用miRanda和TargetScan软件进行差异表达的mmu-miR-671-5p靶基因的预测,预测的靶基因分别有11 953和9 252个,将预测结果取交集进行韦恩(Venn)分析,重叠部分的靶基因有3 681个;利用GO与KEGG进行功能富集性分析,再利用PicTar软件进一步对mmu-miR-671-5p的靶基因进行预测,将预测结果与Venn分析结果进一步取交集,结果显示,mmu-miR-671-5p的预测靶基因有7个;实时荧光定量PCR分别验证7个预测靶基因的相对表达量发现,Tnfrsf1b与Tnip 1基因相对表达量极显著降低(P<0.01);在RAW264.7细胞中转染mmu-miR-671-5p inhibitors,分别验证7个预测靶基因的相对表达量发现,Tnfrsf1b与Tnip 1基因的相对表达量极显著升高(P<0.01);对mmu-miR-671-5p与Tnfrsf1b和Tnip1的3′非翻译区(3′UTR)结合靶位点分别进行预测,结果初步表明,mmu-miR-671-5p的靶基因为Tnfrsf1b与Tnip 1,其预测结合靶位点均只有1个,分别位于Tnfrsf1b和Tnip1 3′UTR全长位置的91和305 bp。本研究结果为进一步揭示mmu-miR-671-5p在布鲁氏菌感染RAW264.7细胞过程中的功能提供科学依据。

In order to predict the target gene of mmu-miR-671-5p mediated by Brucella,the differentially expressed mmu-miRNA-671-5p target genes were predicted by miRanda and TargetScan softwares after RAW264.7 cells were infected by Brucella.The predicted target genes were 11 953 and 9 252,respectively.The predicted results of the two softwares were intersected and analyzed by Venn which showed 3 681 overlapping target genes.The functional enrichment were analyzed by GO and KEGG,and then PicTar software was used to further predict the target gene of mmu-miRNA-671-5p.The predicted results were further intersected with the results of Venn which showed that there were 7 target genes was the finally predicted results of mmu-miRNA-671-5p.The relative expression of 7 predictive target genes was verified by Real-time quantitative PCR.The results showed that the relative expression of Tnfrsf1b and Tnip 1 genes were extremely significantly decreased(P<0.01).And then the mmu-miRNA-671-5p inhibitors were transfected into RAW264.7 cells and the 7 predictive targets were verified.The results showed that the relative expression of Tnfrsf1b and Tnip 1 genes was extremely significantly increased(P<0.01),and the target sites of mmu-miRNA-671-5p and the 3′UTR of Tnfrsf1b and Tnip1 were predicted respectively.The results preliminarily showed that the target genes of mmu-miRNA-671-5p were Tnfrsf1b and Tnip1.Both of them had only one predictive binding site,located at 91 and 305 bp of the full-length positions of 3′UTR of Tnfrsf1b and Tnip1,respectively.This study provided a scientific basis for further revealing the function of mmu-miRNA-671-5p in Brucella infected RAW264.7 cells.