LncRNA H19 Functions As A Competing Endogenous RNA Regulates IGF-1?AR Gene By Targeting MiRNA-29a In PCOS

Objectives:Polycystic ovary syndrome?PCOS?is one of the most common disease gynecological reproductive and endocrine diseases.The main symptoms include:menstrualthinning,obesity,hyperandrogenism,hyperinsulinemia,hyperluteinization,polycystic ovary et.al.Then,the PCOS is a combination of a series of abnormal signs and has a strong clinical heterogeneity.The etiology of PCOS is complex and has not yet been fully unclear.The present study found that the clinical manifestation of PCOS are closely related to hyperandrogenemia and are regulated by multiple signaling pathways.Among them,the two key genes,AR and IGF-1,have attracted much attention and have been proved to play an important roles in the development of PCOS.long noncoding RNAs?lncRNAs?are a kind of noncoding RNA,which do not specially code for proteins.Research study indicates that lncRNAs played a role in wide variety of diseases.Further,some researchers have done high-throughput lncRNAs sequencing in ovarian granulosa cells of patients with PCOS,which showed that three were the significance differencescompared with women.But the function of lncRNAs is largely unclear in PCOS.So,we speculated whether lncRNA is involved in regulating target genes AR and IGF-1 by miRNA in PCOS.H19 is the earliest one of the most extensive lncRNAs research,which has played an important roles in tumor and endocrine disease.It plays an important role in tumor and endocrine diseases.miRNA-29a is a member of the miRNA family and has been reported to correlate with the expression of AR and IGF-1.However,there is currently no report on the relationship between H19 and miRNA-29a in the development of PCOS.Considering the PCOS as a systemic disease,the expression of H19 and miRNA-29a will be detected in peripheral blood of PCOS patients.Considering that the main damage to PCOS is infertility caused by non-ovulation of the ovary,together with the difficulty of acquirement of human ovarian tissue,therefore,we further investigated the relationship between H19 and miRNA-29a in ovarian granulosa cells?KGN?and their effects on downstream genes AR and IGF-1.We preliminary explored the role of H19 in PCOS and its potential as a target gene for diagnosing and treating PCOS in the future.Method:1.59 patients with PCOS diagnosed by Rotterdam and 63 healthy control women were selected,and laboratory indexes in the blood samples were tested.Then we compared the baseline data,basic measurement index,hormone level and metabolic level among the two groups.2.We used real-time fluorescence quantitative polymerase chain reaction?QRT-PCR?to detect the expression of H19 and miRNA-29a by calculating 2^-??CT among 20PCOS patients and 20 healthy controls.3.The biological information forecasting tools?starBase TargetMiner TargetScan and miRcode,etc.?were used to forecast H19 and miRNA-29a,miRNA-29a and AR,binding sites of IGF-1 gene.According to the predicted sites,we constructed that wily and mutant type gene?include:H19,miRNA-29a,AR,IGF-1?vector plasmid.The dual luciferase was used to validate the relationships between H19 and miRNA-29a,miRNA-29a combined with AR and IGF-1 ability in KGN cells;4.The expression of H19 gene and plasmid of siRNA plasmid,miRNA-29a vector plasmid and H19 negative control vector plasmid.NCsiRNA were successfully constructed.5.The mRNA expressions of H19,miRNA-29a,AR,IGF-1 were detect by using qRT-PCR in KGN cells after transferring H19 over-expression plasmid,H19siRNA,miRNA-29a plasmid,alone or in combination.6.The protein expressions of AR and IGF-1 were detected by using Western Blotting in KGN cells after transferring H19 over-expression plasmid,H19siRNA,miRNA-29a plasmid,alone or in combination.,7.SPSS13.0 statistical software and office Excel tables are used for data processing.Analysis of variance was used to measure data,multiple means comparisons.The t-test was used to compare the mean of the two samples,expressed as the mean±standard deviation.Chi-square test was used for count data.The test level is P<0.05.Results:1.The statistical differences were found in BMI,T,P,LH,LH/FSH,AMH,AST,FINS,FPG,HOMA-IR between PCOS group and control group?P<0.05?.2.The expression level of LncRNA H19 of peripheral blood of PCOS patientswas higher than that of the control group,and the difference was statistically significant?P<0.05?;While,the expression of miRNA-29a from peripheral blood of PCOS patients was lower than that of the control group,and the difference was statistically significant?P<0.05?.3.The results of double luciferase reporter geneshowed that the 3'-UTR sequence of LncRNA H19,AR and IGF-1 contained the binding sites of miRNA-29a.QRT-PCR experiments confirmed that miRNA-29a can negatively regulate LncRNA H19,AR and IGF-1.4.H19 gene overexpression vector plasmid,siRNA,miRNA-29a and H19 negative control vector plasmid NCsiRNA were successfully constructed.5.After transferring H19 over-expression vector plasmid into the KGN cells the expression level of H19 gene was increased?vs control group,P<0.05?and the expression of miRNA-29a was decreased?vs control group,P<0.05?,and the expression of AR and IGF-1 was increased?vs control group,P<0.05?.After siRNA was transfected,the expression of H19 gene was decreased?vs control group,P<0.05?,indicating that H19siRNA successfully interfered with the expression of H19,and the expression level of miRNA-29a gene was increased?vs control group,P<0.05?and correspondingly the gene expression levels of AR and IGF-1 were decreased?vs control group,P<0.05?.There were no differences in the expression of H19,miRNA-29a,AR and IGF-1 genes in control group,compared with NCsiRNA and pcDNA 3.1+empty vector group.6.That the expression of AR protein was increased after transfecting H19overexpression vector plasmid into KGN cells compared with blank KGN cells?P<0.05?and the expression of IGF-1 protein increased?P<0.05?.After transfecting H19siRNA,AR protein expression was also increased?vs control group,P<0.05?,and the expression of IGF-1 protein was increased?vs control group,P<0.05?.Compared with NC siRNA and pcDNA 3.1+empty vector group,the expression of H19,miRNA-29a,AR and IGF-1 protein in the control group was not statistically significant.Conclusion:1.Among PCOS patients,androgen levels and insulin are elevated,and the endocrine and metabolic system is disordered.2.The expression of LncRNA H19 in peripheral blood of PCOS patientswas increased?P<0.05?,and the expression of miRNA-29a was decreased?P<0.05?.It is suggested that the LncRNA H19-miRNA-29a may play a role in the pathogenesis of systemic endocrine and metabolic disorders in PCOS,but the specific mechanism still needs further study.3.Double luciferase results showed that H19 and miRNA-29a,miRNA-29a and AR,IGF-1 can affect the dual luciferase fluorescence activity through the 3?UTR binding gene.It was suggested that H19 and miRNA-29a,miRNA-29a and AR,IGF-1 may interact with each other in KGN through the"sponge"adsorption and binding method.4.LncRNA H19 overexpression and H19 siRNA vector plasmids were constructed and transfected into KGN cells.The results showed that the overexpression of LncRNA H19 in vivo may down-regulate the expression of miRNA-29a gene and further regulate the expression of AR and IGF-1 protein,thereby increasing the androgen action in PCOS patients.