LncRNA MEG3 Mediates The Interation Between Apoptosis And Autophagy In The Study Of Spermatogenesis In Non-obstructive Azoospermia

Infertility is one of the medical problems in today’s society.About 15%of couples in the world are affected by infertility,and infertility related to male factors accounts for 30%-55%.72%of male infertility cases are unexplained or idiopathic,and the molecular system behind these defects is still unclear:Non-obstructive azoospermia(NOA)has an incidence of 0.6%in general men,accounting for about 10%of male infertility.Its etiology is unknown,its pathogenesis is complex,and its treatment is still unclear.Although testicular sperm aspiration(TESA)is the standard treatment for NOA,about 50%of patients fail to get sperm by dry testicular puncture.Therefore,it is a challenge to study the molecular mechanism of spermatogenesis and find effective diagnostic markers or therapeutic targets for NOA diagnosis and treatment in clinic.Long non-coding RNA(LncRNA)is a group of regulatory non-protein-coding RNA with more than 200 nucleotides in length,accounting for 80%of non-coding RNA.It regulates basic biochemical and cellular processes through a variety of mechanisms,such as transcription,post-transcriptional modification and epigenetic level.Changes in LncRNA affect different stages of cellular processes,such as cell proliferation,invasion,migration,apoptosis,and autophagy.Studies have found that lncRNA participates in apoptosis and autophagy,and mediates the interaction between autophagy and apoptosis,and participates in apoptosis or autophagy.Poly-ADP-ribose polymerase(PARP)is an important polymerase involved in DNA repair and gene integrity monitoring.By binding Zn2+to eukaryotic DNA,the polymerase can specifically recognize and bind to DNA break ends.At the early stage of the apoptosis process,caspase-3 acts on PARP and cleaved the full-length PARP into 35kda and 89kda fragments to convert PARP to cleaved PARP,in which the 89kda cleaved PARP fragment is generated and then travels out from the nucleus.Therefore,89 kda can be used as an indicator for the detection of cell apoptosis.Caspase-3 gene encodes a protein with a molecular weight of 35kda.When stimulated by apoptosis signal,Caspase cascade reaction is activated,and the full-length caspase-3 is cut into 35kda and 17kda fragments.Activation of caspase-3 to cleaved caspase-3 of 17 kda eventually leads to apoptosis.Relevant studies have shown that MEG3 can regulate the proliferation,apoptosis and autophagy of glioma cells.It has also been reported that lncRNA AK12348 participated in the regulation of myocardial ischemia-reperfusion injury by targeting PARP and Caspase-3.ATG7 gene encodes a protein with a molecular weight of 77kda and plays an important role in the formation of autophagic lysosomes.Beclinl gene encodes a protein with a molecular weight of 60kda.LC3 is mainly involved in the formation of autophagosomes.The LC3 precursor molecule is cleaved by ATG4B to form the cytoplasmic form LC3 I,which is then activated by APG7L/ATG7.It is transferred to ATG3 and coupled to fatty acyl ethanolamine(PE)to form the membrane-bound form LC3 II,which can be attached to the membrane of autophagosome,a structural protein of autophagosome.LC3 I encodes a protein with a molecular weight of 17kda and LC3 II encodes a protein with a molecular weight of 15kda.In recent years,the research on the interaction between apoptosis and autophagy mediated by MEG3 mostly focuses on cancer,but there are few reports on non-obstructive azoospermia.In the previous research of our group,we found that MEG3 was highly expressed in NOA tissues by high-throughput sequencing,and this discovery has been verified by experiments.The results of previous experiments suggest that miR-188-3p/MLH1 mediates apoptosis to regulate NOA spermatogenesis,and miR-188-3p/ATG7 mediates autophagy to regulate NOA spermatogenesis.double luciferase experiment suggests that MEG3 and miR-188-3p have binding sites.The results suggest that MEG3 may mediate the interaction between apoptosis and autophagy and participate in the process of NOA spermatogenesis.Combined with the relevant research progress,this study puts forward a reasonable hypothesis:MEG3 may mediate the interaction between apoptosis and autophagy and participate in the spermatogenesis of non-obstructive azoospermia.Purpose:In this study,qRT-PCR western blot,immunofluorescence and other molecular biological techniques were used to detect the changes of apoptosis and autophagy phenotype after overexpression of MEG3,and to study the function of MEG3.Over-expression of MEG3 and inhibition of autophagy key gene ATG7 were used to detect the changes of apoptosis-related proteins after inhibition of autophagy,to study the influence of autophagy phenotype on apoptosis phenotype,and to explore the relationship between interaction between apoptosis and autophagy mediated by MEG3 and spermatogenesis.Method:1.Experimental grouping:Cell experiments were divided into a steady-state MEG3 group and a steady-state corresponding(negative control)NC group.The cells were transfected with different concentrations of viral vector to determine the optimal concentration for transfection.2.To study the effect of MEG3 on apoptosis:NTERA-2 cells were transfected with MEG3 steady-state virus and the corresponding negative control substance NC to verify the steady-state transformation efficiency.The expression levels of PARP,cleaved PARP,Caspase-3 and cleaved Caspase-3 in two groups were studied by qRT-PCR and western blot experiments.3.Study on the effect of MEG3 on autophagy:NTERA-2 cells were transfected with the MEG3 stably transmitted virus and the corresponding negative control substance NC.The expression levels of Beclinl and LC3 in two groups were studied by qRT-PCR and western blot experiments.Experimental study on the dot-like aggregation of LC3 in cells by cellular immunofluorescence.4.The interaction between apoptosis and autophagy after the expression of MEG3 was studied:NTERA-2 cells were transfected by constructing MEG3 stably transfected virus and the corresponding negative control substance NC,and the transfection efficiency was verified after the autophagy key gene ATG7 was momentarily knocked down.Western blot was used to detect the expressions of apoptosis-related proteins PARP,cleaved PARP,Caspase-3,cleaved Caspase-3,autophagy-related proteins Beclinl and LC3.Result:1.The transfection efficiency of MEG3 was verified after the virus over expression of different MOIs.qRT-PCR results showed that the mRNA expression levels of MEG3 in the groups with MOI 10,50 and 100 were increased.Compared with the control group,the difference was statistically significant(p<0.01).There was no significant difference between groups of viruses that over expressed different MOI.2.After MEG3 over expression,the mRNA expression of Caspase-3 detected by qRT-PCR was increased,and the difference was statistically significant as compared with that in the control group(p<0.05);After the MEG3 was over expressed,the expression levels of apoptosis-related proteins cleaved Caspase-3 and cleaved PARP were detected by western blot.The results showed that the protein expression levels of cleaved Caspase-3 and cleaved PARP were increased compared with the control group.3.After MEG3 over expression,the mRNA expression level of autophagy-related factor Beclinl was detected by qRT-PCR,and the difference was statistically significant as compared with that in the control group(p<0.05).The mRNA expression level of autophagy-related factor LC3 detected by qRT-PCR was significantly different from that in the control group(p<0.001).The protein expression levels of autophagy-related proteins Beclinl and LC3 Ⅱ were detected by western blot,and the results showed that the protein expression levels of Beclinl and LC3 II were increased.Cellular immunofluorescence experiments showed that the degree of fluorescence punctiform aggregation of LC3 over expressed in the MEG3 group was increased compared with the control group.4.After over expressing MEG3,siATG7 was transiently transferred to verify the transfection efficiency,and the mRNA expression of detected ATG7 was significantly reduced,with a statistically significant difference as compared with that in the control group(p<0,01).Over expression of MEG3 was accompanied by knocking down the autophagy key gene ATG7.Western blot results showed that compared with the control group,the protein expression levels of Beclinl and LC3-Ⅱ were decreased.Over expression of MEG3 was accompanied by knockdown of the autophagy key gene ATG7.Western blot assay results showed that compared with the control group,the protein expression levels of cleaved PARP and cleaved Caspase-3 were decreased.Conclusion:1.After over expression of MEG3,the expression levels of genes and proteins related to apoptosis and autophagy are increased,suggesting that MEG3 may be involved in the NOA spermatogenic process by promoting apoptosis and autophagy.2.Knocking down the autophagy key gene ATG7 after over expression of MEG3 inhibits autophagy,and the expression of autophagy-related proteins and apoptosis-related proteins is also reduced,suggesting that MEG3 may be involved in the NOA spermatogenic process by mediating the interaction of apoptosis and autophagy.