Analysis Of MiRNAs-mRNAs Involved In Decidual Function Of Mice And The Role Of Mmu-miR-21a In Regulating Decidual Cells

ObjectiveThe decidualization of endometrium and normal function is the key to a successful pregnancy.Dysfunction of decidualization would lead to placental dysplasia,abortion,and other adverse pregnancy outcomes.Both the transformation of stromal cells into decidual cells and the apoptosis of decidual cells are in a dynamic balance in order to maintain the function of decidual tissue during early pregnancy.This process involves a complex molecular regulatory network.MicroRNA has been confirmed to participate in the decidualization of stromal cells,while its role in decidual cells regulation has been rarely reported,and the specific molecular regulatory mechanism remains unclear.To fully understand the role of microRNA and its target regulated mRNA network in maintaining the function of decidual cells,and to find out the miRNA and its target genes,which play a key role in maintaining the function of decidual tissue,we performed this study.The data could provide an experimental basis for the study of the molecular mechanism of regulating the decidual tissuefunction,and provide clues for the clinical diagnosis and treatment of pregnancy disorders related to decidual abnormalities.Methods1.Establishment of animal model and sample collectionThe sexually mature Kunming female mice and male mice with vasectomy of the same strain were caged at 18:00 p.m.,and documented as day 1 of pseudopregnancy?PD1?if a vaginal plug was investigated the next morning.Artificial decidualization was performed on the fourth day of pseudopregnancy?PD4?.The mice were decapitated and killed at 8:00 a.m.three days after the operation?PD7?,and the appearance of the uterus was observed,photographed.The wet weight of the bilateral uterine horn was recorded,and the tissues were frozen in liquid nitrogen for future use.The expression of Dtprp,a marker of decidualization in mouse endometrium,was detected by fluorescence quantitative PCR.2.Analysis of microRNA and mRNA expression profilesThe next generation sequencing?NGS?was used to analyze the microRNA and mRNA expression profiles in tissue samples.The sequencing work was completed by BGI?Shenzhen,China?.3.Sequencing data processing and bioinformatics analysis?1?The expression profiles of miRNA and mRNA of mice before and after induced decidualization were obtained by NGS.After processing the original data,the known miRNAs and mRNAs with differentexpressions between the control horn and deciduoma were screened out.The expressions of miRNAs and mRNAs in the uterus of the control side and the induced decidualization side of mice were verified by fluorescence quantitative PCR.?2?Targetscan?http://www.targetscan.org/vert₇2/?and microT-CDS?http://diana.imis.athena-innovation.gr/DianaTools/index.php?r=site/page&view=software?were used to predict the potential target mRNAs of differentially expressed miRNAs.Go enrichment and KEGG pathway analysis were performed on the selected miRNAs and their target mRNAs.?3?Go enrichment analysis were performed on the selected mRNAs by NGS.?4?The predicted mRNAs and differentially expressed mRNAs screened by NGS were intersected,and the overlapped mRNAs was used as the potential target genes of miRNAs.Negative regulatory network analysis was then performed.?5?Mutiple predictions were performed on the small RNAs?sRNA?without annotation in the database?Novell miRNA analysis?,including novel miRNA statistics,different expression analysis of novel miRNA,target gene prediction,GO enrichment and KEGG pathway analysis of differential novel miRNA.4.Focus on the mmu-miR-21a-Pdcd4 of negative expressed miRNAs-mRNAs,and the expression of mmu-miR-21 a in the mousedeciduoma module was verified by fluorescence quantitative PCR.5.The primary mouse endometrial stromal cells were isolated,and then the purity of these cells was identified by vimentin and keratin.Estrogen progesterone was induced in the in vitro decidualization.The expression of mmu-miR-21 a was verified by fluorescence quantitative PCR in vitro decidualization model.After the decidual cells were transfected with mmu-miR-21 a inhibitor,the mRNA levels of Dtprp were detected by fluorescence quantitative PCR,and cell apoptosis was detected by flow cytometry.The apoptosis-related proteins and signal pathways were detected by Western blot.6.After the decidual cells were transfected with mmu-miR-21 a inhibitor,the protein levels of PDCD4 were detected by Western blot.The role of mmu-miR-21 a in regulating the expression of PDCD4 in decidual cells was analyzed.The target binding relationship between PDCD4 and mmu-miR-21 a was verified by double luciferase reporter gene detection.7.The mRNA levels of hsa-miR-21 and PDCD4 in human endometrium and decidua were detected by fluorescence quantitative PCR.The human endometrial stromal cell line?CRL-4003?was induced decidualization in vitro,and the decidual cells were transfected with hsa-miR-21 inhibitor.The regulation of hsa-miR-21 on Pdcd4 in human decidual cells was verified.Results1.The results of uterine appearance showed that the induced side was significantly swollen compared with the control side,and its wet weight was significantly increased.The mRNA level of Dtprp,the mouse endometrial decidualization marker,was significantly up-regulated.These data confirmed the successful establishment of the artificially induced decidualization in vivo.2.The expression profiles of miRNA and mRNA of mouse uterus were constructed by NGS technology.The results showed that there were significant differences in the expression of miRNA and mRNA between the control horn and the deciduoma.3.?1?The processing result of original miRNA sequencing data showed that: the high-quality sequencing data are all up to 100%,and more than 90% of the sRNA length is within the range of 20nt-25 nt.The expression of common sequence and specific sequence in the deciduoma was 3.23% and 66.58%,respectively.Compared with the control side,60 miRNAs were up-regulated in the deciduoma?Fold change ?2?,and 34 miRNAs were down-regulated?Fold change ?2?.The results of mRNA sequencing showed that 2346 mRNA were up-regulated?Fold change ?2?,and 3775 mRNA were down-regulated?Fold change ?2?.The results of the quantitative PCR were consistent with the sequencing results.?2?Based on the analysis of the target gene prediction database,10061 target mRNAs of 60 up-regulated miRNAs and 7897 miRNAs target mRNAs of 34 down-regulated miRNAs were obtained.The target genes for the prediction of miRNAs were analyzed again,and the conditions were as follows: miRNA difference ? 2 times,p.value < 0.05 and miT G.score ?0.995.A total of 335 target mRNAs of up-regulated miRNAs and 660 target mRNAs of down-regulated miRNAs were enrolled.GO enrichment analysis of selected miRNAs and their target mRNAs showed that functional clustering was mainly related to biological regulation,cell process,and metabolic regulation.KEGG pathway analysis showed that it was mainly related to metabolism,DNA signal transduction,and JAK-STAT signal pathway.?3?GO enrichment analysis of differently expressed mRNAs showed that functional clustering was mainly related to biological regulation,cell process,and metabolic regulation.?4?The accuracy of the target genes was significantly improved after the intersection of the predicted mRNA,and the differentially expressed mRNAs screened by NGS.The negative regulatory network analysis showed that miRNAs-mRNAs might be involved in the regulation of cell cycle and apoptosis of decidual cells.?5?The results of novel miRNA analysis showed that there were 4miRNAs without annotated in databases,three of which were up-regulated?novel_mir₁,novel_mir₃,novel_mir₄?and one down-regulated?novel_mir₇?in the deciduoma.The results of GO enrichment and KEGG analysis showed that novel miRNA was mainly involved in the biological regulation,cell process,and metabolic signaling pathway.4.The results of fluorescence quantitative PCR showed that the expression of mmu-miR-21 a increased significantly in the deciduoma of mice.5.The results of immunofluorescence of vimentin and keratin showed that the number of cells with positive vimentin and negative keratin was more than 95%,which confirmed the high purity of primary mouse endometrial stromal cells.The expression of mmu-miR-21 a increased significantly after decidualization induced by estrogen and progesterone in vitro.The decidual cells were transfected with mmu-miR-21 a inhibitor,and the levels of mmu-miR-21 a and Dtprp reduced.Flow cytometry showed that the apoptosis of decidual cells increased after inhibition of mmu-miR-21 a.Western blot showed that down-regulation of mmu-miR-21 a could increase the expression of PDCD4,Bax,and Caspase-3 in decidual cells,and significantly reduce the expression of Bcl-2.6.Western blot showed that after inhibiting the expression of mmu-miR-21 a in decidual cells,the protein level of PDCD4 was significantly increased.The results of luciferase reporter gene showed that the luciferase activity of miR-21 co-transfected with wild type PDCD4 wassignificantly lower than that of the control group.In contrast,the luciferase activity of miR-21 co-transfected with mutant PDCD4 had no significant change compared with the control group.7.The fluorescence quantitative PCR results showed that,the expression of PDCD4 in decidual tissues was significantly down-regulated,while the expression of hsa-miR-21 was significantly up-regulated,in decidual tissues,comparing with those in human proliferative endometrium.The human endometrial stromal cells were decidualized in vitro and transfected with hsa-miR-21 inhibitor.The results of flow cytometry showed that the apoptosis of decidual cells was significantly increased in response to the reduction of hsa-miR-21.Conclusion1.The expression profiles of miRNA and mRNA in endometrium before and after decidualization were analyzed by NGS,and the results showed that there were many miRNAs with significant expression differences and their potential target genes.The data suggested that a complex regulatory network of miRNAs-mRNAs participated in the process of decidualization.2.The regulatory network of miRNAs-mRNAs is mainly related to biological regulation,cell process metabolic regulation,and related signaling pathways.In additionally,some microRNAs without functional annotation in the database are also involved in the regulation ofendometrial decidualization.3.MiR-21 can inhibit the apoptosis of decidual cells by target PDCD4 inhibiting the apoptosis of decidual cells,thus maintaining the function of decidual cells during normal pregnancy.Moreover,the regulating function of miR-21 is highly conservative in humans and mice.