The Regulatory Mechanism Of MIR-15a Targeting BDNF/TrkB In Follicular Development And Atresia In Pig

In mammalian animals,there are numbers of follicles growing in the o'varian during each estrus cycle.However,only a few follicles can mature to ovulate,and most of follicles undergo degeneration and are cleared away.The nature of follicle atresia is apoptosis of granulose cell,therefore it is important to inhibite the apoptosis of ovarian granulosa cell for increasing animal reproductive performance.BDNF was firstly isolated and purified from porcine brain in 1982 by Barde et al.Early research about BDNF focused on neuronal systems such as differentiation,proliferation and maturation in nervous system disease.BDNF also plays an important role in reproductive system,such as estrogen secretion,discharge of the first polar body,oocyte maturation and ovulation.MiRNAs are a class of small noncoding regulatory RNAs(19-25 nucleotides)that regulate gene expression posttranscriptionally,involving every cellular process,such as cell growth,differentiation and apoptosis.However,few research about miR-15a and BDNF is investigated in porcine follicular development and atresia.This study,the expression of BDNF was investigated in healthy and atretic follicles with different sizes by immunohistochemistry and qPCR.And we found that the expression of BDNF is highter in medium healthy follicles than that in large healthy and medium atresia follicles.BDNF may be a target gene of miR-15a via some database.We detected BDNF gene expression and protein level in follicle granulose cell after overexpression and inhibition of miR-15a by using qPCR and Western-blotting.It has been reported in some research that p53 can up-regulated expression of miR-15a and estrogen inhibits apotosis of granulose cell.We detected difference of miR-15a and BDNF after stimulation of estrogen.We studied PCNA protein level treated with BDNF.Therefore,we attempt to explore the mechanism of miR-15a to regulate follicular development and atresia,and the results as follows:(1)The highest immunoreactivity of BDNF was found in medium healthy follicles and lower in small and large healthy follicles by immunohistochemistry.In atresia follicle,the level of BDNF mRNA was higher,however,the level of BDNF protein was lower in medium size;the expression of TrkB gene and protein were higher than other sizes.(2)The follicular granulosa cell was extracted from medium,large healthy and medium atresia follicle.The expression of miR-15a was detected by real-time PCR,and found miR-15a was higher in medium atresia follicles and lower in healthy follicles.(3)The follicular granulosa cell from medium healthy follicles were isolated and cultured and treated with doxorubicin.The cell was collected and the expression of p53 and miR-15a were detected by real-time PCR.The result showed that p53 mRNA was significantly increased as well as miR-15a(p<0.05).(4)The expression of miR-15a was significantly down-regulated treated with estrogen(p<0.05).(5)The level of BDNF was increased significantly treated with estrogen.(6)Overexpression of miR-15a could significantly upregulate the expression of BDNF's gene(p<0.05),and inhibition of miR-15a could significantly downregulate the expression of BDNF gene(p<0.05).However,the change of BDNF protein presented the inverse trend.(7)The gene's expression and protein of PCNA was increased significantly treated with BDNF(p<0.05).