Study On MiR-29c-5p Regulating The Functional Status Of Buffalo Follicular Granulosa Cells By Targeting INHBA

Follicular granulosa cells are important functional cells in the follicles,and the changes of their functional states such as apoptosis,proliferation,and steroid hormone secretion are closely related to the development.MicroRNA(miRNA)is a kind of small non-coding RNA molecule,which is widely involved in many physiological processes such as apoptosis,proliferation,autophagy,steroid hormone synthesis of granulosa cells.According to the previous high-flux sequencing of buffalo healthy and atretic follicular granulosa cells,miR-29c-5p was found to be highly expressed in buffalo atretic follicles,suggesting that it may be involved in the regulation of buffalo atretic follicles.This study used techniques such as qRT-PCR,Western blot、ELISA,flow cytometry,EdU,and combined with gene overexpression,interference,and co-transfection methods to explore the mechanism of miR-29c-5p and its target genes in the regulation of the functional status of buffalo follicular granulosa cells,which will provide a theoretical basis for in-depth study of the regulatory mechanism of buffalo follicular development and atresia and improve buffalo reproductive efficiency.1.miR-29c-5p regulates the functional status of buffalo follicular granulosa cells.The effects of miR-29c-5p on the proliferation,apoptosis and steroid hormone synthesis of buffalo granulosa cells were studied.The results showed that the expression of miR-29c-5p in granulosa cells of buffalo atresia follicles was higher than that in healthy follicles(P<0.01).miR-29c-5p mimics(mimics)and miR-29c-5p inhibitor(inhibitor)were transfected into granulosa cells.The detection results of EdU,flow cytometry and ELISA show that mimics can significantly inhibit the proliferation of buffalo granular cells(P<0.05),promote the apoptosis(P<0.01),and inhibit the secretion of steroid hormones of estradiol(E2)and progesterone(PROG)(P<0.01).QRT-PCR and Western blot results showed that the changes in the relative expression levels of genes or proteins related to proliferation,apoptosis and steroid hormone synthesis were consistent with the changes in cell phenotype.The results of inhibitor treatment were the opposite of the mimics results.2.miR-29c-5p regulates the functional state of buffalo follicular granulosa cells by targeting INHBA gene.The results of bioinformatics analysis,qRT-PCR,and double luciferase reporter assay showed that INHBA was the target gene of miR-29c-5p;Overexpression of INHBA significantly promoted the proliferation of buffalo follicular granulosa cells,inhibited cell apoptosis,and promoted the secretion of steroid hormones of E2 and PROG(P<0.01).The changes of relative expression levels of genes related to proliferation,apoptosis and steroid hormone synthesis were consistent with the changes of cell phenotype.The miR-29c-5p mimics is co-transfected with an INHBA overexpression vector,and the results show that INHBA reverses the regulation effect of miR-29c-5p on the functional state of buffalo granulosa cells,further indicating that miR-29c-5p regulates the functional state of buffalo granulosa cells through a target gene INHBA.3.To study the mechanism of INHBA regulating the functional status of buffalo follicle granulosa cells.The PA subunit encoded by INHBA is a component of activin A(ACV-A)and inhibin A(INH-A).It was further verified that INHBA could regulate the functional state of buffalo follicular granulosa cells by increasing the secretion of ACV-A and INH-A.The results showed that miR-29c-5p mimics significantly reduced the secretion of AC V-A and INH-A of buffalo follicle granulosa cells cultured in vitro,while the overexpression of INHBA significantly increased the secretion of ACV-A and INH-A(P<0.01).The buffalo granulosa cells were treated with different concentrations(0,1,10,and 100 ng/mL)of ACV-A or INH-A recombinant protein,and it was found that ACV-A(10 and 100 ng/mL)and INH-A(100 ng/mL)promoted the proliferation and inhibited the apoptosis of buffalo granulosa cells,as well as increased the expression of genes related to proliferation,apoptosis inhibition and steroid hormone synthesis,and reduced the expression of genes related to pro-apoptosis(P<0.05).The above results indicated that miR-29c-5p promoted the apoptosis and inhibited the proliferation of buffalo follicular granulosa cells as well as the synthesis of steroid hormones of E2 and PROG by targeting INHBA gene.INHBA gene regulates the functional state of buffalo follicular granulosa cells by promoting the production of AC V-A and INH-A.