Mir-26a Regulates Apoptosis Of Porcine Ovarian Granulosa Cells By Targeting DHCR24 And Smad4

Ovary is an important reproductive organ in mammals,and its ovulation and regulation are closely related to reproductive performance.The proliferation,apoptosis and functional differentiation of ovarian granulosa cells are of great significance to ovary and are also necessary for follicular maturation.At present,many studies have shown that miRNAs and related genes play an important role in regulating the function of granulosa cells.In this study,we used the transcriptome and miRNA sequencing analysis of porcine luteal phase and follicular phase,and screened the key miRNA and mRNA that might affect the transition from luteal phase to follicular phase,and screened candidate gene miR-26a.In this study,Annexin V-FITC/PI flow cytometry,qPCR,Western blot and ELISA were used to study the effect of miR-26a on the function of porcine ovarian granulosa cells and the function of target genes.The binding sites of miR-26a to DHCR24 and Smad4 of target genes were verified by dual luciferase reporter gene system.The aim of this study is to elucidate the function and mechanism of miR-26a in porcine ovarian granulosa cells,and in order to understand the apoptotic mechanism of porcine granulosa cells and lay a foundation for elucidating the genetic regulation of reproductive endocrinology and studying follicular atresia.The specific results of this study are as follows:1.Annexin V-FITC/PI flow cytometry showed that miR-26a could promote the apoptosis of porcine ovarian granulosa cells.The results of ELISA showed that miR-26a could inhibit the secretion of estradiol and progesterone.2.Bioinformatics analysis revealed that microRNA-26a was highly conserved in different species.Bioinformatics was then used to predict the binding sites of microRNA-26a to DHCR24 and Smad4.The results of double luciferase reporter gene system showed that miR-26a could target DHCR24 and Smad4 3'UTR regions to inhibit the fluorescence activity of the reporter gene.The results of qPCR and Western blot showed that miR-26a negatively regulated the expression of DHCR24 and Smad4 at transcriptional and translation levels.3.The results of Annexin V-FITC/PI flow cytometry showed that interference of DHCR24 and Smad4 expression(transfection of si-DHCR24 and si-Smad4)could promote the apoptosis of porcine granulosa cells.ELISA results showed that interfering with DHCR24 expression inhibited the secretion of estradiol and progesterone,and interfering with Smad4 expression promoted the secretion of estradiol and progesterone.These results suggest that miR-26a can promote the apoptosis of porcine granulosa cells by targeting the inhibition of DHCR24 and Smad4 expression,and may inhibit the secretion of estradiol and progesterone by inhibiting the expression of DHCR24 in the steroid pathway.