Study On The Regulation Mechanism Of MiR-101-3p In Ovarian Granulosa Cells Of Dairy Goats Through Target Gene STC1

MicroRNAs is a series of conserved single-strand non-coding RNA molecules,which exist widely in various parts of the organism and participate in the regulation of growth,development,apoptosis and other physiological processes.Among the lots of miRNAs,miR-101-3p is a tumor suppressor involved in many tumor-related biological processes,including proliferation,apoptosis,angiogenesis,drug resistance,invasion and metastasis.Previous laboratory studies had shown that miR-101-3p was differentially expressed in the ovary of dairy goats with single litter size and multiple litter sizes,indicating that miR-101-3p had important regulatory effects on follicular growth and development.However,there are few reports on how miR-101-3p and its target genes regulate the proliferation,apoptosis and secretion of ovarian granulosa cells.Therefore,this experiment used granulosa cells from the ovary of Guanzhong dairy goats as the research object,using high-throughput sequencing,ELISA,CCK-8,EdU,flow cytometry,fluorescence probe in situhybridization,immunohistochemistry,HEstainingandother methods.Interference,overexpression and co-expression methods were used to study the regulation mechanism of miR-101-3p on hormone secretion,cell proliferation and apoptosis of granulosa cells through target gene STC1.The main results are as follows:1.Transcriptome sequencing of overexpressing miR-101-3p in ovarian granulosa cellsOverexpression of miR-101-3p in granulosa cells,142 differentially expressed miRNAs were obtained by high-throughput sequencing and bioinformatics analysis.GO and KEGG enrichment analysis obtained signal pathways that differentially regulate follicular development,such as protein digestion and absorption,cell adhesion molecules（CAMs）and the like.The 10 genes with the highest expression were screened by real-time quantitative PCR.The results showed that the expressions of STC1,NDUFA4L2,C4BPA,FSHR,CNNM1 and GUCY2C were significantly different and consistent with the results of RNA-seq.The expressions of LOC102189835,CLEC9A,KRT7 and LOC102185049 were not significantly different but consistent with the trend of RNA-seq results.2.Regulatory effect of miR-101-3p on proliferation,apoptosis and secretion of ovarian granulosa cellsmiR-101-3p was expressed in 11 tissues of the hypothalamus,muscle,ovary,breast,kidney,uterus,lung,intestine,liver,heart and pituitary of dairy goats,with the highest expression in the lung and the lowest expression in the heart,there was also a higher expression in the ovary.miR-101-3p was expressed in large follicles（>3mm）,small follicles（1-3mm）and corpus luteum,and the expression levels of small follicles and corpus luteum were higher.miR-101-3p promoted the secretion of estrogen（E2）and progesterone（P4）in granulosa cells through regulating the expression of STAR,CYP11A1,CYP19A1,and3β-HSD steroid hormones genesThe results of CCK8,EdU,flow cytometry,cell cycle and apoptosis showed that overexpression of miR-101-3p could promote granulosa cell apoptosis and inhibit cell proliferation.In addition,inhibition of miR-101-3p could promote granulosa cell proliferation and inhibit cells apoptosis.At the mRNA and protein levels,the effects of miR-101-3p on cell proliferation-related genes CCND1,CCNE1,CDK4,PCNA and apoptosis-related genes BAX,BCL2,P53,and Caspase 3 were consistent with the changes of granulosa cell proliferation and apoptosis.miR-101-3p could regulat granulosa cell proliferation and granulosa cell apoptosis through inhibiting phosphorylation levels of key genes PI3K,PTEN,AKT and mTOR in the PI3K/AKT pathway.3.The regulation mechanism of miR-101-3p on granulosa cells through target gene STC1STC1 was expressed in 11 tissues including the hypothalamus,muscle,ovary,breast,kidney,uterus,lung,intestine,liver,heart and pituitary of dairy goats.There were the highest expression in the uterus and the lowest expression in the pituitary,STC1 in the ovary was also a relatively higher expression.STC1 was expressed in large follicles higher than small follicles and corpus luteum.The dual luciferase reporter vector,real-time quantitative and western blot results indicated that STC1 was the target gene of miR-101-3p.The overexpression,interference and co-expression methods were used to verify that miR-101-3p could regulat the expression of STAR,CYP11A1,CYP19A1,and 3β-HSD steroidogenic hormones through the target gene STC1 to promote the secretion of E₂ and P₄ in granulosa cells.The results of CCK8,EdU,flow cytometry,cell cycle and apoptosis showed that miR-101-3p regulated cell proliferation-related genes CCND1,CCNE1,CDK4,and apoptosis-related genes BAX,BCL2,P53,and Caspase 3 through the target gene STC1 to promote granulosa cell apoptosis and inhibit cell proliferation.miR-101-3p could inhibit granulosa cell proliferation and apoptosis through the target gene STC1 by inhibiting the phosphorylation levels of the PI3K/AKT pathway key genes PI3K,PTEN,AKT and mTOR.4.Effect of miR-101-3p and its target gene STC1 on ovarian development in miceFluorescent probe in situ hybridization revealed that miR-101-3p was highly expressed in primordial follicles and ovarian corpus luteum in mice without treatment,indicating that miR-101-3p regulated follicular development and ovulation.In the overexpression group,miR-101-3p is expressed in most regions of the ovary,including membrane cells of primordial follicles,secondary follicles,growing follicles,mature follicles,membrane cells after ovulation,the corpus luteum,stroma cells formed by granulosa cells after ovulation.In the inhibition group,miR-101-3p was hardly expressed in the ovary,and only a small amount of fluorescence was observed in the local interstitial cells.This results indicated that the agonists and antagonists of miR-101-3p were highly efficient and could effectively study the role of miR-101-3p in the ovary.Immunohistochemistry showed that overexpression of miR-101-3p and interference with STC1 significantly inhibited the expression of STC1 in the ovary,and inhibition of miR-101-3p significantly increased STC1 expression.The experimental group can effectively study the role of STC1 in the ovary.From HE staining of mouse ovary,Ovarian individuals small and stunted,the number of antral follicles significantly decreasing,and follicular developing abnormal were found between Overexpression of miR-101-3p and STC1 interference groups.In addition,inhibition of miR-101-3p expression significantly increased the number of follicles and normal ovarian development.The development of mouse follicles is consistent with the miR-101-3p promoting granulosa cell apoptosis and inhibition of proliferation in vitro.