The Study About LncRNA MEG3 On Mechanism Of Insulin Resistance Of Polycystic Ovary Syndrome

Polycystic ovary syndrome(PCOS)is a relatively common reproductive endocrine and metabolic disorder syndrome in women of childbearing age.It is a common cause of anovulatory infertility.The incidence rate of PCOS is 6%to 8%.PCOS patients are highly heterogeneous and can be complicated with menstrual disorder,infertility,obesity,insulin resistance(IR)and metabolic syndrome.IR is present among 50%to 70%of women with PCOS,and comparatively,only 10%to 25%among women without PCOS.Insulin is the only hypoglycemic hormone in the body,which can promote the synthesis of glycogen,fat and protein.Insulin is also an important regulator of sex hormone metabolism,which can stimulate luteinizing hormone secretion,promote follicular membrane cell proliferation,increase androgen synthesis,recruit a large number of immature follicles and inhibit the formation of mature follicles.IR refers to the decrease of insulin sensitivity or the decrease of peripheral tissue response to hormone regulation,resulting in the increase of insulin in serum to maintain normal blood glucose level.The homeostasis model assessment of IR(HOMA-IR)is widely used by scholars at home and abroad to evaluate the degree of IR,which is calculated by the values of fasting blood glucose(FPG)and fasting insulin(FINS).Body mass index(BMI)is an international index used to measure the degree of adult health and obesity,which is calculated by the value of height and weight.Research shows that HOMA-IR and BMI are closely related.In order to study and improve the pregnancy success rate of patients with IR undergoing assisted reproduction,the first part of our study retrospectively collected the clinical data of PCOS and non PCOS patients with normal BMI who underwent in vitro fertilization and embryo transfer(IVF-ET)for the first time,The corresponding statistical analysis was carried out to find the appropriate values of HOMA-IR and BMI to improve the pregnancy rate.In the second part,by establishing PCOS-IR rat model and insulin resistant KGN cell model and using interference technology,we aimed to study the mechanism of long non coding ribonucleic acid maternally expressed gene 3(lncRNA MEG3)and insulin like growth factor 2 binding protein 2(IGF2BP2)in PCOS insulin resistance,provide new therapeutic targets and approaches for PCOS insulin resistance patients,and provide new ideas and directions for improving clinical pregnancy rate.Section One:HOMA-IR for Predicting Clinical Pregnancy Rate during IVF[Objective]1.To determine the cut-off value of insulin resistance index(HOMA-IR)for predicting the clinical pregnancy rate of polycystic ovary syndrome(PCOS)patients and non PCOS patients with normal body mass index(BMI)infertility who underwent first in vitro fertilization and embryo transfer(IVF-ET).2.To analyze the relationship between insulin resistance index(HOMA-IR)and clinical pregnancy rate in patients with PCOS and non PCOS at different body mass index(BMI).[Methods]The data was retrospectively analyzed from 329 normal-weight(BMI<25 kg/m2)and aged 21-40 years women who received first IVF-ET during the period from December 2018 to June 2019.The patients were divided into PCOS group(n=115)and non PCOS group(n=214)according to the 2018 Chinese PCOS diagnostic criteria.We assessed the associations between HOMA-IR and clinical pregnancy rates during IVF in the women with or without PCOS according to different BMI ranges.[Results]1.Compared to non PCOS group,PCOS group had higher levels of BMI,anti-Miillerian hormone(AMH),dehydroepiandrosterone sulfate(DHEAS),luteinizing hormone(LH),testosterone(T),fasting insulin(FINS),triglyceride(TG),total cholesterol(TC),low density lipoprotein(LDL-C),HOMA-IR,antral follicle count(AFC)and estradiol on the day of HCG(E2),biochemical pregnancy rate(p<0.05);they also had a significantly larger number of dominant follicles on the day of HCG,more retrieved oocytes,fertilized oocytes and high-quality embryos(p<0.05);they also had significantly lower total dosage of gonadotropin(Gn),endometrial thickness on the day of HCG,and numbers of transferred embryos(p<0.05).2.Univariate analysis showed that BMI,LH,FINS and HOMA-IR were correlated with clinical pregnancy rate in non PCOS group(p<0.05);FINS,HOMA-IR and systolic blood pressure(SBP)were correlated with clinical pregnancy rate in PCOS group(p<0.05).Multivariate analysis showed that HOMA-IR was significantly correlated with clinical pregnancy rate after adjusting BMI,LH and FINS in non PCOS group(OR=0.045,95%CI:0.005-0.386,p<0.05).3.In non PCOS,clinical pregnancy rate was significantly decreased at the HOMA-IR values ranging from 2.2 to 3.15(OR=0.188,95%CI:0.084-0.42,p<0.05),and at HOMA-IR≥3.15(OR=0.018,95%CI:0.004-0.081,p<0.05).In PCOS,clinical pregnancy rate significantly decreased at the HOMA-IR>3.15(OR=0.15,95%CI:0.044-0.507,p<0.05).4.In non PCOS with BMI<21.45 kg/m2,clinical pregnancy rate was decreased with HOMA-IR≥2.2,and a significant cut-off point at HOMA-IR≥3.15(OR=0.028,95%CI:0.003-0.234,p<0.05);with 21.45≤BMI<25 kg/m2,clinical pregnancy rate was declined significantly at the HOMA-IR≥1.56(OR=0.196,95%CI:0.055-0.704,p<0.05).In PCOS with BMI<21.45 kg/m2,clinical pregnancy rate was decreased as the HOMA-IR increased,but there was no significant cut-off point;with 21.45≤BMI<25 kg/m2,clinical pregnancy rate was declined significantly at the HOMA-IR≥3.15(OR=0.186,95%CI:0.04-0.872,p<0.05).[Conclusion]HOMA-IR and BMI had adverse effects on the IVF outcome of infertility women.Moreover,higher BMI can increase the degree of insulin resistance in infertility women.These findings suggested that only better HOMA-IR and BMI will lead to better IVF results.Section Two:LncRNA MEG3 is Involved in the Proliferation and Apoptosis of Insulin Resistant Granulosa Cells of PCOS by Binding to IGF2BP2[Objective]Long non coding ribonucleic acid maternally expressed gene 3(lncRNA MEG3)has been shown to correlate to hepatic insulin resistance(IR),but the underlying mechanism of LncRNA MEG3 in polycystic ovary syndrome insulin resistance(PCOS-IR)granulosa cells remains unclear.In this study,we investigated the role of LncRNA MEG3 and its potential interaction with insulin like growth factor 2 binding protein 2(IGF2BP2)in IR granulosa cells in order to provide new therapeutic targets and pathways for PCOS-IR patients.[Methods]1.Ten Sprague Dawley(SD)rats were randomly selected and subcutaneously injected with dehydroepiandrosterone(DHEA)6mg/100g every day and fed with high-fat diet to establish PCOS-IR rat model;ten rats in the control group were fed with normal diet.The PCOS-IR rat model was constructed,HE staining was used to detect the expression of the pathological morphology of ovarian tissue,and the ELISA kit was used to detect the expression of follicle stimulating hormone(FSH),luteinizing hormone(LH),and testosterone(T),fasting plasma glucose(FPG),fasting insulin(FINS)in rat serum.2.The the KGN cell model of IR was constructed.After the KGN cell model of IR were interfered with short hairpin RNA(shRNA)-lncRNA MEG3,qRT-PCR was used to detect the expression level of lncRNA MEG3.CCK-8 was used to detect cell viability,and TUNEL kit was used to detect the level of cell apoptosis.The combination of lncRNA MEG3 and IGF2BP2 was predicted by starbase database and verified by RNA immunoprecipitation(RIP)technology and luciferase assay.Western blot was used to detect the expression of IGF2BP2 and apoptosis-related proteins(Bcl-2,Survivin and Bax).[Results]1.The expression of LncRNA MEG3 was higher in the serum and tissues of PCOS-IR rats than control group(p<0.001);In the KGN cell model of IR,the expression of lncRNA MEG3 was higher than control group(p<0.001).2.After IR KGN cells were interfered with shRNA-lncRNA MEG3,the activity of KGN cells model of IR was increased and the apoptosis was inhibited than control group(p<0.001).3.In the tissue of PCOS-IR rats and the KGN cell model of IR,the expression of IGF2BP2 was higher than control group(p<0.001).4.LncRNA MEG3 was mainly distributed in the cytoplasm and acted in combination with IGF2BP2.IGF2BP2 was lower in IR+shRNA-lncRNA MEG3 group than IR group(p<0.001).5.Overexpression of IGF2BP2 partially blocked the effect of interfering lncRNA MEG3 on proliferation and apoptosis of the KGN cell model of IR.[Conclusion]It was confirmed by PCOS-IR animal model and IR KGN model that LncRNA MEG3 is involved in the proliferation and apoptosis of IR granulosa cells by binding to IGF2BP2.The regulation of their expression might provide a new direction for treating PCOS and IR.