Study Of MiR-21-3p On Regulation And Mechanism Of Autophagy In Bovine Ovarian Granulosa Cells

It was shown that about 99% of the follicules in bovine ovary fail to ovulate because of atretic degeneration,which is the main reason of inefficient reproduction and wasted genetic resources in bovine.Micro RNAs are a class of post-transcriptional non-coding small molecules that usually bind to the 3’-UTR region of the target gene,causing degradation of the target gene or inhibiting mRNA translation.Studies have shown that autophagy was regulated by a variety of miRNAs,but it is not clear how miRNAs regulate autophagy in bovine ovarian granulosa cells(BGCs).In this study,we used transcriptome sequencing on the granulosa cells in bovine follicles with different diameters,and it was found that miR-21-3p was specifically expressed in the granulosa cells of bovine follicles with different diameters.In order to analyze the effects of miR-21-3p on autophagy of bovine ovarian granulosa cells,the plasmids of miR-21-3p mimicand inhibitors were constructed and were transfected into BGCs,and the regulatory mechanism of miR-21-3p on autophagy of BGCs was explored.The main results are as follows:1.Transcriptome sequencing was used to measure granulosa cells in different diameters of bovine follicles,and then analyzed the expression of autophagy marker genes in BGCs with different diameters.The results showed that the expression levels of autophagy marker genes LC3,BECN-1and ATG3 increased with the increase of follicle diameter,while the expression levels of P62 decreased with the increase of follicle diameter.At the same time,we found miRNAs specifically expressed and autophagy-related in bovine follicular granulosa cells of different diameters,and found that miR-21-3p expressed in BGCs of different diameters,and its expression gradually decreased with the increase of follicular diameter.2.qRT-PCR and Western blot were used to detect the effects of miR-21-3p on autophagy of BGCs.The results show that,after overexpression miR-21-3p the cell autophagy marker genes LC3、BECN-1、ATG3、ATG5 and ATG7 mRNA expression level and LC3 protein expression were significantly lower than control group(P < 0.05),P62 mRNA and protein expression levels were significantly higher than that of control group(P < 0.05),indicating that miR-21-3p expression inhibits BGCs autophagy marker genes LC3、BECN-1、ATG3、ATG5 and ATG7 expression BGCs;Compared with NC group,the inhibition group LC3、BECN-1、ATG3、ATG5 and ATG7 mRNA expression level and quantity of Lc3 protein expression were significantly higher than that of control group(P < 0.05),P62 mRNA and protein expression levels were significantly lower than the control group(P < 0.05),suggesting that suppression miR-21-3p promoted the cell autophagy marker gene LC3、BECN-1、ATG3、ATG5 and ATG7 expression in BGCs.3.RNAhybrid and Target Scan 7.1 were used to predict the target genes of mir-21-3p,and according to the predicted results,it was found that miR-21-3p targeted the key genes VEGFA and FGF2 in the PI3K/AKT pathway.Furthermore,miR-21-3p was verified to bind to VEGFA 3’UTR and FGF2 3’UTR to inhibit reporter expression in 293 T cells by dual luciferin reporter.Meanwhile,in bovine ovarian granulosa cells,the results of qRT-PCR and Western blot showed that miR-21-3p could negatively regulate VEGFA and FGF2 at the post-transcriptional level,and negatively regulate VEGFA and FGF2 protein expression at the translation level.4.Rescue experiments demonstrated that VEGFA and FGF2,as direct targets of downstream functions of mir-21-3p,affect autophagy of BGCs.The results of the rescue experiment showed that the co-transfection of miR-21-3p inhibitor with si-VEGFA and si-FGF2 into BGCs could could partially save the occurrence of cell autophagy.5.Western blot was used to investigate the effect of miR-21-3p on autophagy signaling pathway by targeting VEGFA and FGF2.The results showed that after overexpression of miR-21-3p,the phosphorylation level of AKT protein was significantly lower than that of the control group(P < 0.05),indicating that overexpression miR-21-3p inhibited autophagy by reducing the phosphorylation level of PI3K/AKT pathway in granulosa cells.Compared with the control group,the phosphorylation level of AKT protein in the inhibition group was significantly higher than that in the control group(P < 0.05),indicating that inhibition of the expression of miR-21-3p in BGCs could promote the autophagy by activating the phosphorylation level of the PI3K/AKT pathway.6.qRT-PCR and radioimmunoassay were used to detect the effect of miR-21-3p on granulosa cells steroid hormone production.The results showed that transfection of miR-21-3p mimicsignificantly increased the content of E2 in the medium,while inhibition of miR-21-3p decreased the production of E2.Transfection of miR-21-3p mimicfailed to affect the production of cell culture medium P4 in BGCs,but inhibition of miR-21-3p significantly increased the content of P4 in the culture medium.At the same time,the expression of CYP19A1 mRNA in BGCs after transfection with miR-21-3p mimicwas significantly increased,while the expression of CYP19A1 mRNA in BGCs after transfection with mir-21-3p inhibitor was significantly decreased.3βHSD trend is the same as the trend of CYP19A1 express,but after the miR-21-3p 3βHSD expressions had no change.In summary,this study remained that(1)Autophagy can occur in different diameters of BGCs.(2)miR-21-3p can regulate autophagy of BGCs;(3)miR-21-3p regulates the PI3K/Akt pathway and steroid hormone production by targeting VEGFA and FGF2.The results of this study provide theoretical support for the discussion of autophagy of bovine ovarian granulosa cells.