Study On The MiR-188-3p/ATG7 Mediated Autophagy In Spermatogenesis Of Non-obstructive Azoospermia

Non-obstructive azoospermia(NOA)also known as testicular failure,which has a testicular spermatogenic dysfunction that results in the inability to produce sperm or produce a very few amount of sperm.It is reported that NOA patients account for 6% ~ 26% of male infertility.China has a large population,and the number of NOA patients is large,and the number is increasing year by year.However,the pathogenesis of NOA is complex,and the specific mechanism is still unclear.In particular,the molecular mechanism that determines the amount of sperm in NOA patients needs to be further clarified.In recent years,many new technologies have been introduced for the treatment of NOA,but the overall cure rate is still low.Therefore,intensive study on the molecular mechanism affecting the spermatogenesis of NOA patients will help to find new molecular markers for NOA diagnosis and gene therapy targets.MicroRNA(miRNA)is a new type of single-stranded small-molecule RNA of about 22 nucleotides in length.Studies have confirmed that some miRNAs can regulate the expression of target genes and participate in a variety of physiological and pathological processes.Studies in some animals have found that abnormal expression of miR-188-3p leads to abnormal germ cell development and cause sperm formation disorders.Our previous study of this subject also found that miR-188-3p regulates the target gene MLH1 and causes the apoptosis of spermatogenic cells to affect the occurrence of NOA.All of the above studies suggest that miR-188-3p is involved in spermatogenesis.In recent years,more and more studies have shown that miRNAs can regulate autophagy by regulating the expression of their target genes.The autophagy is still the focus of current research in the field of scientific.Autophagy is the use of lysosomes to degrade the damaged organelles and macromolecules under the regulation of autophagy-related genes,and participate in the development of various diseases.ATG7 is a member of autophagy-related genes,which is crucial for the formation of autophagy coupling systems.A study have found that ATG7 knockout mice have acrosome development defects,and the shape of the sperm formed is similar to human round head sperm.Our previous studies found that ATG7 was highly expressed in NOA testis tissue,and miR-188-3p was negatively correlated with ATG7 expression.Further bioinformatics prediction analysis revealed that miR-188-3p and ATG7 have binding sites.However,whether miR-188-3p and ATG7 have direct targeting regulation affect spermatogenesis that further verification is needed.LC3 and Beclin-1 act as autophagy related genes and play important roles in autophagy.Studies have shown that LC3-II is often used as a marker of intracellular autophagy during autophagosome formation.Beclin-1 is a key regulator of autophagy.Overexpression of Beclin-1 activates autophagy,and sufficient level of Beclin-1 is necessary for its autophagy function.It has been reported that miR-30 targets binding to Beclin-1 to downregulate its expression and inhibit autophagy in neuromyeloma cell lines.It has also been reported that miR-21 is upregulated in chronic myelogenous leukemia,and treatment with anti-miR-21 can increase the expression of autophagy-related proteins Beclin-1 and LC3-II,resulting in increased autophagy.Combined with the above research progress,this study proposed the hypothesis that down-regulated miR-188-3p could increase the expression of ATG7 and participate in the process of autophagy by reducing the inhibitory effect on target gene ATG7,so that abnormal increase of autophagy level in testicular tissues of NOA patients would result in decreased spermatogenic function.Purpose:Based on the previous studies,we need to further identify the binding sites and interactions relationship between miR-188-3p and ATG7,and through the detection of autophagy marker gene LC3 and Beclin-1.To investigate whether miR-188-3p affects the level of autophagy by regulating ATG7 and thus affects the occurrence of NOA,and lays a theoretical foundation for further study on the mechanism of NOA.Method:1.MiR-188-3p mimic/mimic NC were transfected NTERA-2 cells,and ATG7 wild type and mutant plasmid of 3’UTR were builded,and the relationship between mir-188-3p and ATG7 was verified by experimental methods such as co-transfection and double fluorescence enzyme reporting gene.2.Experimental group: testicular tissues of non-obstructive azoospermia group and normal control group,the expression levels of autophagy markers LC3 and Beclin-1 genes in the two groups were detected by qRT-PCR.The expression levels of LC3 and Beclin-1 protein in the two groups were detected by Western Blot.The expression and location of LC3 in the testis tissues of the two groups was identified by immunohistochemistry.3.Groups were divided into miR-188-3p mimic /mimic NC and miR-188-3p inhibitor /inhibitor NC.Four groups were transfected into NTERA-2 cells,respectively.qRT-PCR and Western Blot were used to detect the expression of LC3 and Beclin-1 in NTERA-2 cells.The degree of dot-like aggregation of LC3 protein in NTERA-2 cells was identified by immunofluorescence technique.The number of autophagosomes in NTERA-2 cells was detected by electron microscopy.Results:1.The results of double fluorescein enzyme experiment showed that the luciferase activity of ATG7 3’UTR-WT are lower than that of negative control group after overexpression of miR-188-3p(P<0.05),the ATG7 3’UTR-MUT luciferase activity is not significantly different from the negative control group(P>0.05).2.The expression levels of autophagy markers LC3 and Beclin-1 are higher in NOA testis than in normal testis(P<0.05).Immunohistochemistry showed that LC3 is mainly localized in the cytoplasm of spermatogenic cells,and the expression of LC3 protein in NOA group is higher than that in the control group(P<0.05).3.After overexpression of miR-188-3p in NTERA-2 cells,the expression levels of LC3 and Beclin-1 are lower than those of the none overexpression group(P<0.05).The expression levels of LC3 and Beclin-1 after inhibition miR-188-3p are higher than that of the none inhibiton group(P<0.05).The results of immunofluorescence show that the overexpression of miR-188-3p group has lower dot-like aggregation of LC3 protein than the control group,and inhibition of miR-188-3p group have higher dot-like aggregation of LC3 protein than the control group.The results of transmission electron microscopy show that the number of autophagosomes in the overexpresseion miR-188-3p group is lower than that in the control group.Conclusion:1.The autophagy marker genes LC3 and Beclin-1 are highly expressed in NOA tissues.After overexpression or inhibition of miR-188-3p,the expression of LC3 and Beclin-1 genes is negatively correlated with miR-188-3p expression,indicating that miR-188-3p may be involved the process of autophagy in NOA patients.2.ATG7 is the target gene of miR-188-3p,suggesting that miR-188-3p may regulate the target gene ATG7 to participate in autophagy and affect the development of NOA.