Study On The Mechanism Of LncRNA MEG3/miR-188-3p-mediated Apoptosis Regulating Spermatogenesis In Non-obstructive Azoospermia

Non-obstructive azoospermia(NOA)is considered to be the most serious clinical manifestation of male infertility,also known as testicular spermatogenic failure,affecting about 1%of men and 10%of infertile men.Because the mechanism is unknown and there is no effective targeted treatment,it is a serious disease for men with infertility.The population of non-obstructive azoospermia in China has a large base.At present,for these people,the only way to obtain parental generation is to use intracytoplasmic sperm microinjection((intracytoplasmic sperm injection,ICSI),),but about 50%of the patients are still unable to obtain normal sperm.The research on the mechanism of spermatogenesis in NOA needs to be solved urgently.Further research on the molecular mechanism of sperm failure will help us to provide new ideas for clinical treatment.Long non-coding RNA is a RNA molecule with a length of about 200 nt and 100000 mol/L,which participates in a variety of biological cell regulation processes.Studies have found that IncRNA can participate in apoptosis through the ceRNA mechanism,and then affect classical apoptotic pathways such as p53 to regulate cancer cell apoptosis.In the previous study,through high-throughput sequencing,we found that the expression levels of MEG3 and miR-188-3p in testicular tissue of NOA had significant statistical significance,the experiment confirmed that there was negative regulation between them,and bioinformatics analysis predicted that there were potential binding sites,which suggested that there may be a mechanism of ceRNA action between them.In recent years,the studies on lncRNA MEG3 and p53 are mostly focused on cancer,but there are few reports on non-obstructive azoospermia.In the previous study,we found that p53 gene was highly expressed in testicular tissues of patients with NOA.P53 gene encodes a protein with molecular weight of 43.7KDa,which is classified as a tumor suppressor because the deletion of its wild type affects tumor growth.It has been reported that p53 can participate in the classical apoptosis pathway and induce apoptosis through endogenous apoptosis pathway.Apaf-1 is an important apoptotic factor and participates in the formation of apoptotic bodies.It has been reported that lncRNA MEG3 can regulate the expression of Apaf-1 through ceRNA mechanism and promote the apoptosis of laryngeal cancer cells.Related studies have shown that lncRNA can regulate apoptosis factors through ceRNA,and then affect spermatogenesis in non-obstructive azoospermia.Other studies have reported that lncRNA MEG3 affects p53 and the process of cancer cell apoptosis through ceRNA mechanism in many cancers.Combined with the related research progress,this study proposes the hypothesis that lncRNA MEG3 may regulate the expression of p53 and apaf-1 through the ceRNA mechanism and affect the spermatogenesis of NO A through the apoptotic pathway.Purpose:The binding site of miR-188-3p and lncRNA MEG3 was verified by constructing double luciferase experiment.By interfering with lncRNA MEG3,to detect the expression level of p53 and apaf-1 genes,to explore the gene regulation process of lncRNA MEG3,p53 and apaf-1.The purpose of this study is to make a new interpretation of the mechanism of sperm apoptosis in NOA.Method:1.The expression of autophagy markers p53 and Beclin-1 in Noa testicular tissue and normal functional testicular tissue was studied by means of QRT-PCR and western-blot,and the expression and localization of immunohistochemical methods in testicular tissues with different spermatogenic functions were identified apaf-1.2.To study the effects of mir-188-3p on autophagy,using mir-188-3p simulations(mimic),inhibitors(inhibitor)and corresponding negative controls(negative control)to transfection NTERA-2 cells through QRT-PCR,The expression of autophagy markers apaf-1 and Beclin-1 in various groups of NTERA-2 cells was studied by Western-blot and other methods,and the degree of midpoint aggregation of apaf-1 protein in NTERA-2 cells was identified by immunofluorescence technique.The number of autophagy in NTERA-2 cells in each group was observed by transmission electron microscopy.3.Use miR-188-3p mimic/mimic NC transfection NTERA-2 cells to build p53 Wild type and mutant plasmid of 3’UTR,and the relationship between miR-188-3p and p53 was verified by experimental methods such as co-transfection and double fluorescence enzyme reporting gene.Result:1.Bioinformatics suggests that lncRNA MEG3 is the target gene of miR-188-3p.The results of double fluorescein assay showed that the luciferase activity of lncRNA MEG3 3’UTR-WT after overexpression of miR-188-3p was lower than that of the control group(p<0.05).There was no significant difference in),lncRNA MEG33’UTR-MUT luciferase activity between the control group and the control group(p<0.05).2.qRT-PCR and Western-Blot showed that the expression of p53 and apaf-1 in NO A testicular tissue was higher than that in normal testicular tissue,and the immunofluorescence results showed that the expression of p53 protein and apaf-1 protein were distributed in the nucleus and cytoplasm of NTERA-2.3.After interfering with lncRNA MEG3,the expression levels of p53 and apaf-1 were lower than those in the control group.The results of immunofluorescence showed that the expression of p53 protein decreased in the cytoplasm of the interfering lncRNA MEG3 group,and there was no significant change in apaf-1.The results of transmission electron microscope showed that the number of apoptotic bodies in the interference lncRNA MEG3 group was less than that in the negative control group.The results of apoptosis detected by flow cytometry showed that the number of apoptotic cells decreased in both early and late stages of interference with lncRNA MEG3.Conclusion:1.P53 and apaf-1 were highly expressed in NOA,and the expression of p53 and apaf-1 decreased after interfering with lncRNA MEG3,indicating that lncRNA MEG3 may participate in the process of apoptosis in NOA patients by regulating p53 and apaf-1.2.There are binding sites between miR-188-3p and lncRNA MEG3,and they may have regulatory effects,suggesting that miR-188-3p may affect the level of apoptosis by regulating the target gene lncRNA MEG3,thus participating in the occurrence and development of NOA.