Mechanism Of Clusterin By Regulated METTL3/lncRNA TUG1 Alleviates Diabetic Testicular Injury

Objective:Diabetic testicular dysfunction is an important complication of the reproductive system of diabetic men,which has brought great harm to men’s mental health and reproductive function.However,the mechanism of occurrence and progression of testicular dysfunction in diabetes is still unclear.Clusterin(CLU),also known as apolipoprotein J and secreted by astrocytes,has been reported to be involved in various tumor processes,including tumorigenesis,development,and metastasis.In recent years,a growing number of studies have found that Clusterin plays a critical role in the development of diabetes.For example,Clusterin can affect the incidence of type 2 diabetes or regulate diabetic cerebral infarction and other critical diseases.Taken together,in this study,we selected Clusterin as the research core,and targeted diabetic testicular dysfunction to clarify the biological function of Clusterin in diabetic testicular injury.Clearly,to establish a regulatory network with Clusterin as the core regulator and METTL3 as the upstream regulator to inhibit diabetic testicular injury by mediating LncRNA Tug1-Clusterin signaling axis to deactivate the PI3K/AKT/m TOR signaling pathway and its mediated autophagy.This study uncovers a new mechanism for the occurrence and progression of diabetic testicular damage,and could find new targets for the treatment of diabetic testicular dysfunction.Method:The STZ-induced diabetic rat model was constructed and transfected with Clusterin,METTL3,LncRNA TUG1 overexpression vector,or treated with LY294002.Afterwards,ELISA,IHC,HE staining,TUNEL detection,q PCR and WB were used to detect serum testosterone level,METTL3 expression level in testicular tissue of diabetic rats,testicular tissue injury degree and testicular tissue apoptosis degree of diabetic rats,Clusterin,METTL3,LncRNA TUG1,autophagy related proteins(LC3 II,LC3 I,Beclin-1,p62)and apoptosis related proteins(Bcl-2,Bax,cleaved caspase-3 and cleaved caspase-9)mRNA and protein expression levels,respectively.Next,high glucose was used to construct GC-1 spg cell damage model,and the cells were also transfected with Clusterin,METTL3,LncRNA TUG1,Clusterin overexpression and SRSF1 silencing vectors,or treated with rapamycin and LY294002.MTT assay,flow cytometry,autophagy flow assay,immunofluorescence assay,RT-PCR and WB assay,RNA stability assay,RIP andRNA pull-down assay were used to detect cell viability,apoptosis levels,autophagy levels,the expression level of Clusterin,LncRNA TUG1,autophagy related proteins(LC3II,LC3 I,Beclin-1,P62),apoptosis related proteins(Bcl-2,Bax,cleaved caspase-3 and cleaved caspase-9)and PI3K/Akt/m TOR signaling pathways(PI3K,p-PI3 K,Akt,p-Akt,m TOR,p-m TOR),stability of Clusterin mRNA,and the interaction between TUG1 and Clusterin or METTL3 and TUG1.Result:Effects of Clusterin on autophagy and apoptosis during testicular injury induced by diabetes: In rat model,ELISA showed that the serum testosterone level of STZ-induced animals was significantly lower than that of the control group.The IHC results revealed that the expression of Clusterin in the testis tissue of diabetic rats was significantly lower than that of the control group,while the ratio of LC3 II to LC3 Iwas significantly higher than that of the control group.And the WB further confirmed that STZ could significantly inhibit Clusterin and improve the ratio of LC3 II to LC3 I.In cell models,immunofluorescence detection showed that the expression of Clusterin in the nucleus and cytoplasm was significantly down-regulated after induction by high glucose.In addition,high glucose induction was able to significantly reduce the viability of GC-1 spg cells,promote cell apoptosis,and improve apoptosis-related proteins(Bax,cleaved caspase-3 and cleaved caspase-9)or autophagy-related proteins(ratio of LC3 II to LC3 I and Beclin-1)expression,and simultaneously inhibit the Bcl-2 and p62 expression.Autophagy flux assay found that autophagosomes and autophagolysosomes increased significantly after high glucose induction.But the above indexes could be restored after transfection with Clusterin overexpression vector,while adding rapamycin(RAP)treatment could weaken the recovery effect of Clusterin overexpression.Clusterin regulated diabetes-induced autophagy in rat testis injury by activating PI3K/AKT/m TOR signaling pathway: WB detection showed that the expression levels of p-PI3 K,p-Akt,p-m TOR,and p62 were significantly decreased,and autophagy-related proteins(ratio of LC3 II to LC3 I and Beclin-1)were significantly up-regulated in high glucose-induced GC-1 spg cells,indicating that high glucose inhibited PI3K/AKT/m TOR signaling pathway activation,but activated autophagy.And Clusterin overexpression could activate PI3K/AKT/m TOR signaling pathway to inhibit autophagy,but the effect of Clusterin overexpression could be reversed after LY294002 treatment.Moreover,ELISA results indicated that the testosterone levels of the serum of STZ rats was significantly increased after overexpression of Clusterin.HE staining revealed that compared with the control group,the testicular tissue of STZ rats was severely damaged,and with obvious pathological changes including the diameter of the seminiferous tubules becoming smaller,the arrangement was not compact,and the cavities in the lumen were cavitated.At the same time,it was also observed that the expression of autophagy-related proteins(ratio of LC3 II to LC3 I and Beclin-1)was significantly promoted,and the expression of p62 was significantly inhibited.Furthermore,the results of TUNEL showed that the number of apoptotic spermatogenic cells in the testis tissue of STZ rats was significantly higher than that of the normal control group.Similarly,the above indicators could be improved by Clusterin overexpression,but the addition of LY294002 could weaken the protective effect of Clusterin overexpression.METTL3 enhanced spermatogenic cell viability and inhibited high glucose-induced spermatogenic cell apoptosis: High glucose could significantly reduce the viability of GC-1 spg cells and promote cell apoptosis.Similarly,the protein expressions of Bax,cleaved caspase-3 and cleaved caspase-9 are significantly up-regulated,and Bcl-2 protein expression was down-regulated in high glucose-induced GC-1 spg cells,and overexpression of METTL3 could reverse the above results.METTL3 enhanced the stability of Clusterin and inhibited high glucose-induced spermatogenic cell apoptosis by binding SRSF1 through TUG1: The expression of LncRNA TUG1 was significantly decreased in high glucose-induced GC-1 spg cells.RMVar predicted that there is an m6 A modification site in LncRNA TUG1.RIP confirmed that TUG1 was enriched in the anti-METTL3 antibody complex.In addition,theRNA pull-down results showed that the LncRNA TUG1 mRNA 3’-UTR probe could pull the METTL3 protein,which indicated that METTL3 binds to the 3’-UTR region of LncRNA TUG1.In addition,overexpression of METTL3 was able to significantly increase the expression of TUG1 and decrease the degradation rate of LncRNA TUG1 mRNA in high glucose-induced GC-1 spg cells.However,knockdown of TUG1 could reverse the elevated TUG1 expression caused by overexpression of METTL3.Moreover,overexpression of METTL3 could significantly increase the viability of GC-1 spg cells and decrease the apoptosis rate of GC-1 spg cells,but knockdown of TUG1 could reverse the promotion of cell viability and the inhibition of apoptosis by overexpression of METTL3.In addition,RIP andRNA pull-down assays showed that SRSF1 formed anRNA-protein complex with TUG1.And knockdown of SRSF1 can significantly inhibit the expression of SRSF1 and Clusterin.After overexpression of TUG1,the expression of Clusterin mRNA was up-regulated,but this effect was inhibited by SRSF1 silencing.In addition,RIP results confirmed that overexpression of TUG1 enhanced the abundance of Clusterin in the anti-SRSF1 antibody treated group.Finally,we found that TUG1 up-regulation was also able to significantly enhance the stability of Clusterin mRNA,but this result could be reversed by SRSF1 silencing.Conclusions:1.Clusterin alleviates diabetes-induced testicular damage by inhibiting high glucose-induced autophagy and spermatogenic cell apoptosis.2.Clusterin can activate PI3K/Akt/m TOR signaling pathway to slow down diabetes-induced testicular damage.3.METTL3 regulates LncRNA TUG1 through m6 A modification to play a protective role in high glucose-induced GC-1 spg cells injury.4.LncRNA TUG1 regulates diabetic testicular damage by recruiting SRSF1 to increase the stability of Clusterin mRNA.