Mechanism Of CircINHA Regulating Granulosa Cell Apoptosis By CTGF During Porcine Follicular Atresia

The reproductive potential of female mammals is based on the development and growth of ovarian follicles.However,more than 99%of ovarian follicles undergo atresia,which is a complex and delicate biological process.Ovarian follicular development is closely related to extensive remodeling of the extracellular matrix and angiogenesis,which are also the principal functions of CTGF.Our previous transcriptome analysis showed that CTGF was significatly down regulated during porcine follicular atresia,but its mechanism in porcine follicular development and atresia is not yet clear.Moreover,the emerging of endogenous non-coding RNAs study has provided a new aspect to explore the regulatory mechanisms involved in follicular atresia.circular RNA(circRNA)is a newly discovered ncRNA that regulates gene expression through a variety of mechanisms in mammals.However,its expression and function in porcine ovarian follicles,especially in follicular atresia,remains unclear.In this study,porcine ovarian follicles and follicular granulosa cells(GCs)were used to study the expression characteristics and functions of circRNA during follicular atresia,and then the involvement of circINHA in the expression regulation of CTGF was analyzed in porcine follicular granulosa cells.Our results provided novel insights into the potential mechanism involved in the initiation of follicular atresia in pig ovary.The main results of this study are as follows:1.The expression profiles of circRNAs during porcine follicular atresiaThe deep sequencing of circRNA was performed in healthy and early atretic follicles,and 40567 distinct circRNAs were identified.Further analysis revealed 197 differentially expressed circRNAs during follicular atresia,including 108 upregulated and 89 downregulated circRNAs.Comparison analysis between the expression of mRNA and circRNA host genes highlighted the possibility of a reinforce mechanism of co-transcription of circRNAs and their linear mRNA in inhibin proteins INHBA and INHBB,glutathione S-transferase GSTA1 and vascular endothelial growth factor VEGFA.circRNA-miRNAs interactions prediction provided functional candidates which may play roles in modulating gene expression post transcriptionally via miRNAs.The protein coding potential of circRNAs was also analyzed.The results showed that circRNA is extensivelly expressed in porcine ovarian follicles and plays a potential role in the initiation of follicular atresia.2.CTGF promotes porcine GCs proliferaton and inhibits porcine GCs apoptosisFirstlty.the distribution and expression of CTGF in ovarian follicles were detected by immunohistochemistry and qRT-PCR.The result showed a wide distribution of CTGF in ovarian follicles,especially in GCs,and more expression in healthy follicle(HF)than atresic follicle(AF).CTGF mRNA in theca cells and thecal GCs was significantly higher in HF than AF.Then,recombinant protein and RNA interference(siRNA)were used to overexpress and knock down CTGF in porcine GCs,and analyze its effect on the proliferation and apoptosis of porcine GCs.The results showed that CTGF affected GCs proliferation and apoptosis at a dose dependent manner.CTGF specific siRNA effectively knocked down the expression of CTGF and significantly inhibited porcine GCs proliferation and promoted porcine GCs apoptosis rate.These results indicated that CTGF has the effect of promoting proliferation and anti-apoptosis in porcine GCs.3.miR-10a-5p regulates procine GCs proliferation and apoptosis by via CTGFAccording to our previous results from miRNAs microarray during follicular atresia,miR-10a-5p was selected as a candidate miRNA targeting to CTGF,and qRT-PCR also comfirmed that miR-10a-5p was significantly down-regulated during porcine follicle atresia.The luciferase reporter gene,qRT-PCR and western blot confirmed that miR-10a-5p inhibited CTGF expression by targeting the 3’UTR of CTGF.The CCK-8 confirmed that miR-10a-5p regulates the procine GCs proliferation by targeting CTGF.The FACS confirmed that miR-10a-5p promoted the procine GCs apoptosis via CTGF.These results fully demonstrated that miR-10a-5p targets CTGF to regulate the proliferation and apoptosis of procine GCs.4.circINHA increased CTGF expression by competitive binding miR-10a-5p and regulates procine GCs apoptosisFrom the differentially expressed circRNA,circINHA was selected as the research object for further analysis.The circular structure of circINHA was confirmed by sanger sequencing and qRT-PCR confirmed its down-regulated in the atretic follicle.Then,in procine GCs cultured in vitro,circINHA and miR-10a-5p were directly bound by luciferase activity,RNA pulldown of biotin-labeled miRNA and FISH assay.Finally,the synthesis of circINHA-specific siRNA significantly inhibited circINHA level but did not affect the level of linear INHA.Both mRNA and protein levels of CTGF in procine GCs were reduced by si-circINHA.These effects were reversed by additional miR-10a-5p inhibitor.At the same time,the apoptosis rate of procine GCs increased after transfection of si-circINHA,and this effect was reversed by additional miR-10a-5p inhibitor.Results above indicated that circINHA suppress GCs apoptosis via CTGF by acting as a ceRNA for miR-10a-5p.In summary,our results indicated that circRNAs specifically expressed and had potential functions during porcine ovarian follicular atresia.circINHA inhibited follicular GCs apoptosis by regulating CTGF expression via competitive binding with miR-10a-5p.Our study will add new knowledge for circRNA expression characteristics and functions during early follicular atresia in pig ovary,provide possible biomarkers for researches on ovarian dysfunctions,and provide new insights into the underlying mechanisms in the initiation of porcine ovarian follicular atresia.