Study On The Mechanism Of LncRNA MEG3/miR-188-3p/ATG7 In Regulation Of Non-obsructive Azoospermia

Non-obstructive azoospermia(NOA)is one of the most severe male infertilities,and the pathogenesis is still unclear.Nowadays,along with the development of bioinformatics and microarray analysis technology,research on the function of non-coding RNA(ncRNA)has attracted more and more attentions.NcRNAs are proved to be involved in the development of many diseases.Our previous study showed that the lower expression of miR-188-3p can induce the apotosis of spermatogenic cell through MLH1.Long non-coding RNA is a class of regulatory non-coding RNA,which is longer than 200kb.Researchers found that IncRNA can competitively bind to miRNA to relieve the inhibitory effect of miRNA on mRNA,thereby indirectly regulating the expression of mRNA,to influence the occurrence and development of diseases.Based on the study of miR-188-3p,we firstly identified the IncRNA and mRNA expression profiles in testis tissues of NOA patients and the normal testis tissues through high-throughput sequencing.Furthermore,we identified the potential binding sites between IncRNA MEG3 and miR-188-3p,ATG7 and miR-188-3p through microarray analysis.LncRNA MEG3 is a kind of maternal imprinting gene.The studies about it were mainly on the occurrence of tumor.The lower expression level of IncRNA MEG3 in tumor cells can inhibit the apoptosis of tumor cells.Fewer study on the relationship between IncRNA MEG3 and azoospermia,the function and mechanism are still unclear.ATG7,which is a pivotal gene in cell autophagy,is a kind of mRNA.Researchers found that miR-188-3p could inhibit cell autophagy and affect the progress of cardiac infarction and heart failure through binding to ATG7.And ATG7 is essential for the development of sperm acrosomal.The study about the relationship between ATG7 and azoospermia is still unclear.Based on these findings,we hipothesised that whether lncRNA MEG3 could affect the occurrence of NOA through binding to miR-188-3p competitively,indirectly regulating the expression level of ATG7.Purpose:To verify the results of high-throughput sequencing and investigate the mechanism of lncRNA MEG3 regulating the expression of ATG7 through miR-188-3p,therefore regulate the occurrence of NOA.To provide new insight and theoretical basis for the treatment of NOA.Method:1.Experimental group:Divided testis tissues into NOA group(20 cases)and normal group(patients with normal spermatogenesis function,20 cases).Divided cells into cells with overexpression/suppression of miR-188-3p and cells without overexpression/suppression of miR-188-3p.2.To detect the IncRNA and mRNA expression profiles in NOA group and normal group through,high-throughput sequencing.According to the results of the sequencing,we predicted the lncRNAs and mRNAs which have the binding sites of miR-188-3p with bioinformatics analysis.2.To study the expression of IncRNA MEG3 and ATG7 in different testis tissues by RT-qPCR and western-blot.Identify the localization of IncRNA MEG3 in testis and NTERA-2 cells by in situ hybridization.Identify the localization of ATG7 in testis by immunohistochemistry.3.Through transfection of miR-188-3p mimic,miR-188-3p inhibitor and the negative control to NTERA-2 cells,RT-qPCR and western-blot were used to detect the regulation of IncRNA MEG3 and ATG7 by miR-188-3p.Result:1.The results of high-throughput sequencing showed that total of 122 IncRNAs(53 IncRNAs were up-regulated and 69 IncRNAs were down-regulated)and 109 mRNAs(93 of them were expressed Up-regulated,down-regulated 16 mRNAs)differentially expressed between NOA testis and normal testis;Bioinformatics analysis found that 15 differentially expressed IncRNAs had the binding sites with miR-188-3p,among that IncRNA MEG3 expressed most significantly different;And 5 differentially expressed mRNAs had the binding sites with miR-188-3p,among that ATG7 was a critical gene in the process of cell autophagy.2.Compared with normal group,the expression levels of IncRNA MEG3 and ATG7 in NOA group significantly increased(P<0.05).3.IncRNA MEG3 localized in the cytoplasm and nuclear of all kinds cells in testis;IncRNA MEG3 localized in the cytoplasm and nuclear of NTERA-2 cell,mainly in cytoplasm.ATG7 localized in the cytoplasm of spermatogenic cells;4.After overexpressing miR-188-3p,the expression levels of IncRNA MEG3 and ATG7 are significantly lower than those of control(P<0.05).After inhibiting miR-188-3p,the expression levels of IncRNA MEG3 and ATG7 are significantly higher than those of control(P<0.05).Conclusion:1.The overexpression and location of IncRNA MEG3 and ATG7 may influence the development of NOA.2.The negative relationship between IncRNA MEG3,ATG7 and miR-188-3p,suggesting that IncRNA MEG3 may involve in the regulation of ATG7 through miR-188-3p.