Identification Of Caprine Rumen Related MiRNAs And Functional Study Of MiR-148a-3p

Rumen is the biggest compartment of stomach in adult ruminant,which plays an important role in digestion of foods and absorption of the nutrients.Also,it provides a place for microbes to ferment.Rumen experiences a period from formation to formation of muscle column,to differentiation of different layer,to differentiation of epithelium.The regulatory mechanisms during this period are seldom reported.MicroRNA(miRNA)participates in organic development,differentiation,proliferation and apoptosis by post-transcriptional regulation.Thus,anlysis on expression of miRNAs during development of rumen in fetal goat can contribute to understand the regulatory machanisms from molecular level.In this study,rumens of embryo stage 60 and 135 day were utilized to high-throughput sequencing.The expression of miRNAs in these rumens and functional anlysis of highly and differentially expressed miRNAs were analyzed,respectively.Furthermore,preliminary analysis of biological roles of miR-148a-3p was performed.The main results are summarized as follows.1.Every sample got a clean data of more than 13.84 M after beening filtered by quality control.Clean data was mapped to reference genome.The result showed that the samples got a percentage of mapped reads of 52.93,57.67,60.35,53.63,51.94 and 55.43,respectively.Among these mapped reads,there were 982 miRNAs,including 432 known miRNAs and 559 novel miRNAs.2.Anlysis on preference of base showed that the first base of miRNAs with a length of 18 nt did not contain cytosine,and adenine and guanine accounted for a large proportion.The first base of miRNAs with a length of 19 nt only contain adenine and uracil,and uracil accounted for a relatively large proportion.MiRNA with a length of 20-25 nt contained all of four bases in the frist position,and adenine and uracil accounted for a relatively large proportion.Cytosine had a relatively small proportion at each position of miRNAs.Adenine also seldom been seen at 5-8 nt and 16-24 nt.Uracil was abundant at most of the positions.3.Analysis on highly expressed miRNAs showed that they were mainly correlative with proliferation,growth and apoptosis.Enrichment analysis found that their target genes mainly participated in the basic reaction of cells and signaling pathways correlative with proliferation,growth and differentiation.Moreover,their target genes were related to cell cycle and apotosis.Analysis on differentially expressed miRNAs showed that except the basic reaction,their target genes also enriched in several transcriptional and regulatory processes.These results indicated that ruminal development had differentially regulation during the period.4.The results of dual luciferase report assay showed that miR-148a-3p regulated the expression of QKI by directly targeting 3'-UTR of QKI.5.Co-location of miR-148a-3p and QKI in rumen,reticulum,omasum,abomasum,duodenal,campylobacter,ileal and colon were detected.Signals of miR-148a-3p and QKI were displayed at the same position.Signal strength at epithelium was much higher than other layers,which means that miR-148a-3p plays important roles in epithelium by regulating the expression of QKI.However,signals in duodenal,campylobacter,ileal and colon located at different places.Moreover,higher signals were found at blood-vessel wall in colon.These results indicated that miR-148a-3p may not have biological function in intestinal tract.However,an unknown function of miR-148a-3p in blood-vessel wall was also indicated.6.Overexpression and knockdown of miR-148a-3p in GES-1 were performed in this study.Overexpression of miR-148a-3p has no significant effect on celluar proliferation.This was probably due to the highly endogenous expression of GES-1.However,knockdown of miR-148a-3p contributed to higher ability of proliferation on 48 h after transfection,which indicated that miR-148a-3p had a similar function in normal gastric epithelial cells in contrast with caner cells.Moreover,the expression of QKI isforms in miR-148a-3p over-expressed and inhibiting cells indicated that the expression of QKI5 had a negative correlation with miR-148a-3p.Combining the result of dual luciferase report assay,we can speculate that QKI5 was directly targeted by miR-148a-3p.