Functional Study Of Lnc-SNX30-2:1 In Ovarian Granulosa Cells Of Infertility Patients With PCOS

Polycystic ovary syndrome(PCOS)is a common gynecological disease with a global prevalence of 4%-21%.PCOS can affect many aspects of women’s overall health and is the most common cause of anovulatory infertility;The incidence rate of gestational diabetes,preeclampsia,fetal macrosomia and perinatal mortality in PCOS patients is significantly higher than that in the control group.In addition,PCOS can affect women’s spirit and emotion.Therefore,the study of the disease characteristics and pathogenesis of PCOS is of great significance for the prevention and treatment of PCOS in China.Previous studies have shown that PCOS is the result of the interaction of genetic,environmental and lifestyle factors.However,there is no universal treatment for PCOS.The etiology of PCOS has not been fully clarified.Ovarian hyperandrogenemia,ovarian follicular development disorder,insulin resistance,neuroendocrine,mitochondrial mutation and dysfunction,genetic variation and so on are considered to be related to PCOS.Long noncoding RNA(LncRNA)is a non-protein coding region transcript of more than 200 nt in length,widely expressed in various organisms including human,regulating tumorigenesis and development,cardiac hypertrophy,Myocardial infarction,heart failure,bone development,bone balance and other pathological processes.Recent studies have shown that lncRNA is involved in the regulation of gynecological diseases such as gynecological tumors and endometriosis.In PCOS patients,some research chip based methods reported and verified the differentially expressed lncRNAs in cumulus cells,granulosa cells,follicular fluid and ovarian tissues,and revealed the functions of some lncRNAs in PCOS.However,there is still a lack of lncRNA expression profile of PCOS patients based on high-throughput sequencing.Follicle is the basic unit of reproductive system.Granulosa cells,as an important cell of follicle,can affect the development of follicle by changing the microenvironment.Because ovarian follicle growth inhibition is one of the characteristics of PCOS,the overall expression profile of lncRNA in granulosa cells under PCOS pathological conditions is still lack of corresponding data;At the same time,the function of lncRNA in PCOS remains to be further studied.Therefore,the study of lncRNA omics information of ovarian granulosa cells may provide an important understanding for clarifying the pathogenesis of PCOS and improving the infertility outcome of PCOS.In this study,we first revealed the lncRNA expression profile in granulosa cells of PCOS patients by high-throughput sequencing,then verified the differentially expressed lncRNAs expression by qPCR,and screened the key molecule lnc-SNX30-2:1,Then,the correlation between lnc-SNX30-2:1 and assisted reproductive outcome was analyzed,and the biological function and clinical significance of lnc-SNX3 0-2:1 were preliminarily discussed by using ovarian granulosa cell line KGN as a model,which provided new insights for clarifying the pathogenesis of PCOS.Part I LncRNA expression profiling of ovarian granulosa cells from infertile patients with PCOSObjectiveTo screen differentially expressed lncRNA in ovarian granulosa cells of PCOS infertility patients with RNA-sequencing(RNA-seq)technique.Method1.Five cases of PCOS infertility patients in reproductive center of Zhengzhou University People’s Hospital were collected as study group and five cases of tubal factor infertility patients in the same period as control.Total RNA was extracted from the 10 patients.The expression of lncRNA in the two groups was compared by RNA-seq method.2.Based on RNA-seq sequencing results,using Cluster thermogram showed the differences of lncRNA expression profiles between granulosa cells of infertility patients in PCOS group(n=5)and control group(n=5),and screened the differentially expressed lncRNA.3.The candidate target genes of differentially expressed lncRNAs were predicted and analyzed,and their function were predicted by GO enrichment analysis,KEGG enrichment analysis.4.LncRNA-miRNA-mRNA interaction network construction were performed to predicte the possible functions and regulatory networks of the differentially expressed lncRNAs.5.The top 10 differentially expressed lncRNAs(four differentially upregulated and two differentially downregulated related to proliferation and apoptosis)were identified using qPCR.Result1.A total of 13867 lncRNAs were detected in RNA-seq.275 differentially expressed lncRNAs were found in PCOS patient group,of which 125 were up-regulated and 150 were down-regulated according to the log2(fold change)≥ 1 and FDR<0.001 criteria.2.GO analysis showed that differentially expressed lncRNAs were mainly distributed in annotations of cellular components and biological process.3.KEGG pathway enrichment analysis showed that differentially expressed lncRNA was mainly enriched in PI3K-AKT,MAPK(mitogen-activated protein kinases,MAPK)and RAS signaling pathways.4.The lncRNA-miRNA-mRNA interaction network revealed differentially expressed lncRNA regulatory networks containing a range of miRNAs and mRNAs involved in cell proliferation and apoptosis.5.Among the top 10 differentially expressed lncRNAs,qPCR results verified that lnc-snx3 0-2:1 related to proliferation and apoptosis was significantly down regulated in ovarian granulosa cells of infertile patients with PCOS.ConclusionThe expression profile of lncRNA in ovarian granulosa cells of infertile patients with PCOS was different from that of normal controls.The target genes of differentially expressed lncRNAs are mainly enriched in PI3K/AKT,MAPK and other signal pathways related to cell proliferation and apoptosis.Lnc-SNX30-2:1 is a candidate key lncRNA related to the proliferation and apoptosis of ovarian granulosa cells in infertile patients with PCOS.Part Ⅱ Functional Analysis of lnc-SNX30-2:1ObjectiveTo investigate the expression characteristics of lnc-SNX30-2:1 in infertile patients with PCOS and its possible clinical correlation.And its biological function was preliminarily explored.Method1.26 patients with PCOS infertility and 29 patients with tubal infertility in the same period who underwent in vitro fertilization embryo transfer in the reproductive center of the people’s Hospital of Zhengzhou University were selected as the research objects.The clinical data of the two groups,hCG day serum and granulosa cells on the day of egg collection,were collected.The clinical data and the expression level of lnc-SNX3 0-2:1 were compared between the two groups.2.Proliferation,cell apoptosis and cell cycle distribution were detected in lnc-SNX30-2:1 overexpressed KGN cells using MTS and flow cytometry.3.Target gene predictions,GO analysis,KEGG analysis of lnc-SNX30-2:1 and lnc-SNX30-2:1-miRNA-mRNA network map were performed to predict its biological functions.Result1.Lnc-SNX30-2:1 was down-regulated in ovarian granulosa cells and serum of PCOS patients by qPCR detection.2.It was negatively correlated with the number of left and right sinusoidal follicles(r=-0.385,P<0.05;r=-0.402,P<0.05)and positively correlated with the daily FSH level of HCG(r=0.352,P<0.05).The total number of eggs and normal cleavage in the high expression group of lnc-SNX30-2:1 were significantly higher than those in the low expression group(18.10±2.865 vs 10.36±1.785,P<0.05;9.727±1.349 vs 5.600±1.439,P<0.05).3.MTS assay and flow cytometry analysis showed that overexpression of lnc-SNX30-2:1 in ovarian granulosa cells promoted cell proliferation,inhibited apoptosis,and promoted G1 to S phase transition.4.Target gene analysis by GO and KEGG lnc-SNX30-2:1 revealed that the regulatory network of lnc-SNX30-2:1 in ovarian granulosa cells was mainly enriched in proliferation,apoptosis,cycle and metabolism related molecules and signaling pathways.ConclusionThe expression of lnc-SNX30-2:1 in serum and ovarian granulosa cells of infertile patients with PCOS was significantly lower than that in normal group.Functional analysis showed that lnc-SNX30-2:1 has the biological functions of promoting granulosa cell proliferation,inhibiting granulosa cell apoptosis and promoting G1 to S phase transformation.It may mediate the occurrence and development of PCOS by regulating granulosa cell proliferation,cycle and apoptosis related signal pathways.