Transcriptome Analysis And Related Pathways Research Of Inflammatory Related MiRNA In Bovine Endometrial Stromal Cells

After delivery,bovine uterine stromal cells are often exposed to complex bacterial and viral stimulation due to epithelial cell rupture,resulting in inflammatory reaction and cow abortion.In order to study the pathogenesis of endometritis,we used an in vitro model to study the response of bovine endometrial stromal cells(b ESCs)to inflammatory mediator lipopolysaccharide(LPS).At the same time,we used high-throughput sequencing technology to detect the miRNAs of normal cells and LPS stimulated cells,and screened the miRNAs with large expression differences for bioinformatics analysis to explore the molecular mechanism of its participation in inflammatory response.Based on the comprehensive analysis results,bta-miR-1247-3p and bta-miR-200 c with large differences in expression were screened for further research.The targeting relationship between bta-miR-1247-3p and bta-miR-200 c and target genes MAPK11 and ZEB1 was verified through double luciferase reporting test.After overexpression of miRNA in b ESCs,the changes of the expression of related molecules were detected to explore the involvement of bta-miR-1247-3p and bta-miR-200 c in the regulation of inflammatory response of bovine endometrial stromal cells.This paper provides a new theoretical basis for the pathogenesis of bovine endometritis from the perspective of miRNA.The research contents of this paper include:(1)Isolation and culture of bovine endometrial stromal cells and establishment of in vitro inflammatory modelBovine endometrial stromal cells were isolated and cultured by tissue block culture method.Stromal cells were isolated and purified by trypsin differential digestion method.Stromal cell specific vimentin was identified by immunofluorescence method.The results showed that on the second day,the tissue block adhered to the wall and cells crawled out of the tissue block.On the sixth day,the cell monolayer spread out.After passage to the third generation,epithelial cells and other cells could be removed to obtain stromal cells with high purity.Under the microscope,the cells were spindle shaped,and the immunofluorescence results showed that the expression of vimentin was positive.P3-p8 cells with high purity and good vitality were selected and stimulated with 0.5 μg/ml LPS for 12 hours.The inflammatory factor(IL-1β),TNF-α,IL-6,IL-8 were detected by RT-q PCR.The results showed that the expression of inflammatory factors in LPS treatment group was significantly higher than that in control group,indicating that the in vitro inflammatory model was successfully constructed,which laid the foundation for the next step of high-throughput sequencing.(2)Changes of miRNA expression in bovine endometrial stromal cells stimulated by LPSHigh throughput sequencing was used to identify miRNAs that may have anti-inflammatory effects in lipopolysaccharide induced inflammatory response.BESCs were divided into blank group and LPS treatment group.Compared with the control group,219 miRNAs were differentially expressed in the LPS treatment group,of which113 were up-regulated and 106 were down regulated.Go enrichment analysis showed that the target genes differentially expressed miRNAs were significantly enriched in transferase activity,small molecule binding and protein binding.KEGG pathway analysis showed that the target genes of differential miRNAs were significantly enriched in fluid shear stress and atherosclerosis,MAPK signaling pathway and TNF signaling pathway.By analyzing differentially expressed miRNAs,we found that bta-miR-200 c target genes(ZEB1,ZEB2)and bta-miR-1247-3p target genes(MAPK11,MAPK4,MAPK8,MAPK14)are mainly enriched in transcriptional misregulation in cancer and MAPK pathway,which provides ideas for further verifying miRNA’s participation in inflammatory response.(3)Screening and validation of bta-miR-1247-3p and bta-miR-200 c target genes and their molecular pathwaysUsing the bioinformatics software program targetscan 7.2,the target genes MAPK11 and ZEB1 of bta-miR-1247-3p and bta-miR-200 c were screened.According to the bovine MAPK11 and ZEB1 3?-UTR sequence information published in NCBI website as the template,the fragments containing the corresponding binding sites of bta-miR-1247-3p and bta-miR-200 c were amplified,and then the fragments without their binding sites were amplified with deletion primers.The fragments were inserted into the double fluorescence report plasmid psicheck-2 to construct wild-type and deletion vectors.The plasmids and miRNA mimics were co transfected into b ESCs,and the fluorescence intensity was detected after 24 hours.The results of double fluorescence showed that bta-miR-1247-3p and bta-miR-200 c could significantly reduce the luciferase activity containing their corresponding binding sites.The results of RT-q PCR and Western blot showed that overexpression of bta-miR-1247-3p could reduce the expression of MAPK11 and ATF-2 and reduce the expression of inflammatory factor IL-1β and TNF-α.In addition,overexpression of bta-miR-200 c can reduce ZEB1 and promote the expression of caspase 3 and cleaved caspase-3.It is suggested that overexpression of bta-miR-1247-3p can reduce the inflammatory response of cells,and overexpression of bta-miR-200 c can promote the expression of apoptotic protein.In conclusion,through high-throughput sequencing,it was found that the miRNA expression profile changed after LPS stimulated b ESCs,in which bta-miR-1247-3p could regulate the inflammatory response by targeting MAPK11,and bta-miR-200 c could regulate the expression of apoptotic protein by targeting ZEB1.