lncRNA-MSTRG.7889.1 Competitively Binds to bta-miR-146a Targeting Smad4 to Regulate Apoptosis of Yak Granulosa Cells
[Background]Apoptosis of granulosa cells(GCs)is an important cause of follicular atresia,and competing endogenous RNA mechanism has been proven to be involved in regulating the apoptosis process of GCs.The previous transcriptome sequencing study of yak ovary showed that lncRNA-MSTRG.7889.1 had binding sites with bta-miR-146a,while bta-miR-146a had a targeting relationship with Smad4.[Objective]The aim of this experiment was to explore the molecular regulatory mechanisms of ceRNA that affected the apoptosis of follicular GCs in yak,so as to lay a foundation for revealing the mystery of the reproductive regulation of the yak.[Method]Healthy and atretic follicles of adult female yaks were collected and isolated,and the expressions of lncRNA-MSTRG.7889.1,bta-miR-146a and Smad4 in follicles were detected by RT-qPCR.Healthy and atretic follicles were sliced,and GCs apoptosis in healthy and atretic follicles was detected by TUNEL.The localizations of lncRNA-MSTRG.7889.1,bta-miR-146a and Smad4 in follicles were analyzed by fluorescence in situ hybridization.The yak GCs were isolated and cultured in vitro,and transfected with Smad4 overexpression vector and bta-miR-146a mimics,respectively.The apoptosis rate of GCs cells was detected by flow cytometry,and the expressions of pro-apoptotic proteins CASPASE3 and BAX and anti-apoptotic proteins BCL-2 were detected by Western blot.After co-transfecting Smad4 overexpression vector and bta-miR-146a mimics with GCs,it was investigated whether bta-miR-146a targeting Smad4 affects GCs apoptosis in yaks.lncRNA-MSTRG.7889.1 overexpression lentiviral vector was used to infect GCs,and the effect of lncRNA-MSTRG.7889.1 on GCs apoptosis was detected by flow cytometry and Western blot.lncRNA-MSTRG.7889.1 vector and bta-miR-146a mimics were co-transfected with GCs to further analyze whether lncRNA-MSTRG.7889.1 competitive binding of bta-miR-146a targeting Smad4 affected the apoptosis of GCs in yaks.[Result]The expression of lncRNA-MSTRG.7889.1 and Smad4 gene mRNA in healthy yak follicles was significantly higher than that in atresia follicles(P＜0.01),while the expression of bta-miR-146a was opposite(P＜0.01).TUNEL test results showed that the fluorescence intensity of GCs in atretic follicles was significantly higher than that in healthy follicles(P＜0.01),indicating that the apoptosis of GCs in atretic follicles was significantly higher than that in healthy follicles.The results of fluorescence in situ hybridization showed that Smad4,bta-miR-146a and lncRNA-MSTRG.7889.1 were co-expressed in the follicles of yaks,and their expressions in healthy and atretic follicles were basically consistent with the results of RT-qPCR,indicating that ceRNA mechanism might be involved in the development of healthy follicles and follicle atresia in yaks.Overexpression of Smad4 in yaks GCs significantly decreased the apoptosis rate of GCs(P＜0.01),and the expressions of CASPASE3 and BAX protein were significantly decreased(P＜0.01),while the expression of BCL-2 was significantly increased(P＜0.01).Overexpression of bta-miR-146a significantly increased the apoptosis rate of GCs(P＜0.01),increased the expressions of CASPASE3 and BAX proteins(P＜0.01),and decreased the expression of BCL-2(P＜0.01).bta-miR-146a targeted inhibition of Smad4 expression(P＜0.01);Smad4 and bta-miR-146a were co-transfected into GCs,and the results showed that bta-miR-146a targeted inhibition of Smad4 reduced the former's promoting effect on GCs apoptosis(P＜0.01).Overexpression of lncRNA-MSTRG.7889.1 significantly decreased the apoptosis rate of GCs in yaks(P＜0.01),the expressions of CASPASE3 and BAX protein were significantly decreased(P＜0.01),and the expression of BCL-2 was significantly increased(P＜0.01).lncRNA-MSTRG.7889.1 significantly inhibited the expression of bta-miR-146a(P＜0.01).lncRNA-MSTRG.7889.1 and bta-miR-146a were co-transfected into GCs,and lncRNA-MSTRG.7889.1 targeting bta-miR-146a decreased the promotion effect of the latter on GCs apoptosis(P＜0.01),and the results of RT-qPCR and Western blot showed that lncRNA-MSTRG7889.1 competitively bound bta-miR-146a to promote the expression of Smad4 gene at mRNA and protein levels(P＜0.01).[Conclusion]lncRNA-MSTRG.7889.1 competitively binds to bta-miR-146a,promotes the expression of Smad4 and inhibits the apoptosis of yak GCs.
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