The Correlation Of MiR-362-3p And MiR-361-3p With Missed Abortion And Its Possible Pathogenesis

Objectives:1.To detect the expression of miR-362-3p and its target genes MCAM mRNA,miR-361-3p and its target genes FSTL3 mRNA,SCARB1 mRNA and ITGA5 mRNA in the villi tissues of the artificial abortion group and missed abortion group,and explore their correlation with missed abortion;2.To detect the protein expression of MCAM and CD34 genes in the villi tissues of the artificial abortion group and the missed abortion group,and explore the effect of MCAM on the formation of villi microvessels in the first trimester;3.By analyzing and comparing the correlation between the relative expression level of miR-362-3p and MCAM mRNA in villus tissue,and the correlation between the protein expression level of MCAM and CD34 gene,to brief explored the mechanism of miR-362-3p in the occurrence of missed abortion in early pregnancy.Methods:1.Bioinformatics analysis: miR-362-3p and miR-361-3p whose expression was significantly up-regulated were selected from the whole transcriptome sequencing data of the research group’s early embryonic abortion for analysis and research,and searching for the corresponding target genes.Regarding miR-362-3p target gene prediction,we use the four online websites of Target Scan,ENCORI,micro T-CDS and miRDB to predict its target genes,and use the Venn online website to get the intersection.Select the mRNA that is related to angiogenesis and down-regulated in the previous whole transcriptome sequencing data of our research group,these mRNA and Intersection of the first four database results take the intersection,finally to identify the target gene of miR-362-3p related to angiogenesis-MCAM mRNA.Then use the Target Scan online website to predict the binding site of miR-362-3p and MCAM.Regarding miR-361-3p target gene prediction,we used Target Scan and ENCORI two miRNA target gene prediction websites to predict its target genes,then with down-regulated mRNA related to angiogenesis on the whole transcriptome sequencing of our research group’s previous research,through the Venn online tool to obtain the intersection target gene,combined with literature review,the target genes FSTL3 mRNA,SCARB1 mRNA and ITGA5 mRNA were selected.2.Real-time fluorescence quantitative polymerase chain reaction was used to detect miR-362-3p,miR-361-3p,MCAM mRNA,FSTL3 mRNA,SCARB1 mRNA and ITGA5 mRNA in the villi tissues of 30 patients in the artificial abortion group and 30 patients in the missed abortion group,to analyze the relationship between genes expression and missed abortion.3.Immunohistochemistry was used to detect the protein expression of MCAM and CD34 genes in the villi tissues of 20 cases of the artificial abortion group and 20 cases of missed abortion group,to explore the expression of villi microvessel density.4.Statistical analysis method: SPSS23.0 software was used for statistical analysis.Measurement data were expressed as mean ± standard deviation(`c ±s),and analyzed by independent sample t test.Pearson correlation analysis was used to analyze the correlation between the relative expression level of miR-362-3p and MCAM mRNA,and the correlation between the protein expression level of MCAM and CD34 gene.P <0.05 is considered statistically significantResults:1.According to bioinformatics analysis,regarding of miR-362-3p,we used the four miRNA target gene prediction websites of Target Scan,ENCORI,micro T-CDS,and miRDB to predict that the target genes were 359,3502,1643,and 159,respectively.A total of 35 intersecting target genes were obtained using the Venn online tool,and then 25 mRNAs related to angiogenesis and down-regulated in the whole transcriptome sequencing of our research group’s previous research were intersected to obtain one target gene MCAM;Regarding of miR-361-3p,we used the two miRNA target gene prediction websites of Target Scan,ENCORI to predict that the target genes were 5181 and 2973,respectively,and then 25 mRNAs related to angiogenesis and down-regulated in the whole transcriptome sequencing of our research group’s previous research were intersected to obtain four target genes,combined with literature review,ITGA5,a gene related to angiogenesis,was selected for verification;Among the target genes predicted by the two online websites of Target Scan and ENCORI for miR-361-3p,SCARB1 and FSTL3 were selected for subsequent qRT-PCR verification by consulting the literature and preliminary research.2.The qRT-PCR test results showed that the expression levels of miR-362-3p and miR-361-3p increased in the villi tissues of the missed abortion group,and the difference was statistically significant,P<0.05;the expression level of MCAM mRNA decreased,and the difference was statistically significant,P<0.05;FSTL3 mRNA expression level decreased,and the difference was not statistically significant,P>0.05;SCARB1 mRNA expression level increased,and the difference was statistically significant,P<0.05;ITGA5mRNA expression level increased,and the difference was not statistically significant,P>0.05.3.The results of IHC test showed that MCAM protein was mainly located in the cytoplasm and the intercellular junction of endothelial cells,and the positive staining of MCAM protein was light yellow or brown.The expression level of MCAM protein in the villus tissue of the missed abortion group was lower than that of the artificial abortion group,and the difference was statistically significant,P<0.05;CD34 was localized and expressed in the cytoplasm of vascular endothelial cells,and CD34 positive staining was light yellow or brownish-yellow.The expression level of CD34 protein in villus tissue of the miscarriage group was lower than that of the artificial abortion group,and the difference was statistically significant,P<0.05.4.Pearson correlation analysis results showed that the relative expression level of miR-362-3p was negatively correlated with the relative expression level of MCAM mRNA in the villous tissue of missed abortion,P<0.05;MCAM protein expression was positively correlated with Microvascular Density,P<0.05.Conclusions:1.The expression of miR-361-3p was upregulated in the villous tissue of missed abortion,and the difference was statistically significant(P<0.05),and miR-361-3p is related to the occurrence and development of missed abortion.2.The expression SCARB1 mRNA that is the target gene of miR-361-3p was up-regulated in the villous tissue of missed abortion,and the difference was statistically significant(P<0.05),miR-361-3p may regulate SCARB1 mRNA through acting as an enhancer trigger or other bypasses,thereby related to the occurrence of missed abortion.3.The expression of miR-362-3p was upregulated in the villous tissue of missed abortion,and the difference was statistically significant(P<0.05),and miR-362-3p is related to the occurrence and development of missed abortion.4.The expression of MCAM mRNA,the target gene of miR-362-3p,was down-regulated in the villous tissue of missed abortion,and the difference was statistically significant(P<0.05),miR-362-3p was negatively correlated with the relative expression level of MCAM mRNA,and miR-362-3p may interfere with the formation of villous microvessels by targeting MCAM and then participate in the mechanism of missed abortion in early pregnancy.