The Mechanism Of LncRNA PR11-705C15.3 Regulates Granulosa Cell Autophagy In Polycystic Ovary Syndrome Through The MiR-34c-5p/IGF2 Axis

Objective:Polycystic ovary syndrome is a heterogeneous disease common in women at reproductive age,with the main features of hyperandrogenemia,polycystic ovarian morphology and ovulatory dysfunction.Currently,treatment of PCOS remains a challenge in the clinic.Autophagy is a conserved lysosomal catabolic pathway that provides cells with essential nutrients,and degrades damaged proteins and organelles,which is important for maintaining cellular homeostasis and development.Autophagy is closely regulated by many factors.Insulin-like growth factor 2（IGF2）can through tis receptor of IGF2-R regulate autophagy,and mi R-34c-5p can inhibit autophagy by targeting ATG4B in several types of cells.The aberrant activation of autophagy may be associated with elevated androgen levels,altered folliculogenesis and ovulatory disorders.Our previous study suggested that enhanced autophagy was detected in ovarian tissue of PCOS patients.However,little is known about which factors enhance autophagy in ovarian tissue during PCOS.Long non-coding RNAs（lnc RNAs）are non-coding RNAs of approximately 200nucleotides in length.Functionally,lnc RNAs can act as competing endogenous RNAs（ce RNAs）to sponge its targeted micro RNAs（mi RNAs）to enhance the downstream gene expression or directly interact with some transcription factors to modulate their targeted gene expression.Our previous study has found that lnc RNAs in follicular fluids regulate the development and progression of PCOS in humans.Recently,other studies have shown that lnc RNA modulate sex hormone secretion,the phenotypes of ovary granulosa cells（GCs）and other PCOS-related endocrine metabolism processes.Moreover,lnc RNAs can modulate cell autophagy during the process of other metabolic diseases,such as diabetes and cardiovascular diseases.However,which lnc RNA can regulate autophagy of GCs during the process of PCOS has not been clarified.Our research group discovered and named a lnc RNA RP11-705C15.3 for the first time through RNA sequencing technology in the early stage,the expression of which in PCOS patients was significantly lower than that in normal women,but whether RP11-705C15.3 can regulate the autophagy of GCs during PCOS is still unclear.The main purpose of this study is to deeply reveal the potential important functions of RP11-705C15.3 in the progression of PCOS,and to further explore the molecular mechanism of RP11-705C15.3 as a ce RNA sponge to sponge mi R-34c-5p to enhance IGF2 expression and inhibit GCs autophagy during the process of PCOS.To clarify the effect of autophagy mediated by RP11-705C15.3/mi R-34c-5p/IGF2 axis on the phenotype of PCOS provides new clinical molecular targets and markers for the treatment of PCOS.Methods:1.GCs samples were collected from 20 patients with PCOS and 20 women with normal ovarian function.The expression level of RP11-705C15.3 was detected by RT-PCR.Steroidogenic human ovarian granulose-like tumor KGN and ovarian cancer COV434 cells were obtained from the Cell Bank of the Chinese Academy of Science,and cultured in DMEM/F12 medium containing 10%fetal bovine serum（Gibco,USA）,100 U/ml of penicillin,100μg/ml of streptomycin（Gibco,USA）at 37?°C in an incubator of 5%CO₂.RP11-705C15.3-specific si RNA,negative control si RNA,RP11-705C15.3over-expression plasmid,and control plasmid-NC were obtained from Gene Pharma（China）,and were transfected into KGN and COV434 cells,using Lipofectamine?3000System（Invitrogen,USA）.The efficiency of specific gene silencing or over-expression was verified by q RT-PCR.CCK-8 assay,wound healing assay,Transwell migration assay,and Annexin V/PI Apoptosis assay were utilized to detect the changes of cell proliferation,migration,invasion and apoptosis.Western blot was applied to detect the expressions of autophagy proteins,including LC3-II/I,ATG5,ATG7 and P62.KGN and COV434 cells were co-transduced in triplicate with adenovirus for the expression of m RFP-GFP-LC3（Han Bio,China）,and the fluorescent proteins signals were observed under a fluorescence microscope.The cells were fixed,dehydrated and embedded,examined in transmission electron microscope.2.A dataset was downloaded from GEO database（https://www.ncbi.nlm.nih.gov/geo/）,and the differentially expressed mi RNAs between PCOS and control women by bioinformatics method.RT-PCR was used to detect the expression levels of mi R-34c-5p and IGF2 in GCs samples of 20 pairs of PCOS patients and normal women.Bioinformatics website（https://cm.jefferson.edu/rna22/Interactive/）analysis revealed that the mi R-34c-5p was predicted to have a putative binding sequence with both RP11-705C15.3 and IGF2.Next,we validated the key binding sites by luciferase reporter and RNA pull-down assays.mi R-34c-5p mimics,control mimics,mi R-34c-5p inhibitor,control inhibitor,IGF2-specific si RNA,control scramble si RNA,IGF2 over-expression plasmid,and control plasmid-NC were obtained from Han Bio（China）,and were transfected into KGN and COV434 cells,using Lipofectamine?3000.The efficiency of specific gene silencing or over-expression was verified by q RT-PCR.Western blot was used to detect the expressions of autophagy proteins,including LC3-II/I,ATG5,ATG7and P62.KGN and COV434 cells were co-transduced in triplicate with adenovirus for the expression of m RFP-GFP-LC3,and the fluorescent proteins signals were observed under a fluorescence microscope.The cells were fixed,dehydrated and embedded,examined in transmission electron microscope.3.RP11-705C15.3 and mi R-34c-5p was co-transfected into KGN and COV434 cells via cell transfection method.The expression level of IGF2 was detected by RT-PCR.Moreover,mi R-34c-5p and IGF2,RP11-705C15.3 and IGF2 were co-transfected into KGN and COV434 cells,respectively.Western blot was utilized to detect the expressions of autophagy proteins LC3-II/I,ATG5,ATG7 and P62,and PI3K/Akt signal pathway proteins PI3K,p-PI3K,Akt and p-Akt.KGN and COV434 cells were co-transduced in triplicate with adenovirus for the expression of m RFP-GFP-LC3,and the fluorescent proteins signals were observed under a fluorescence microscope.The cells were fixed,dehydrated and embedded,examined in transmission electron microscope.The PCOS rat models were established by subcutaneous injection of dehydroepiandrosterone（DHEA）.The ovarian morphologies of rats were evaluated via HE staining,and the expression levels of RP11-705C15.3,mi R-34c-5p and IGF2 were detected by RT-PCR.RP11-705C15.3 overexpression and control plasmids were purchased from genepharma（China）.Twenty one PCOS model rats were randomly divided into three groups,including RP11-705C15.3-OE group,RP11-705C15.3-NC group and control group.Seven rats in each group were injected with corresponding plasmid or normal saline through caudal vein,and the transfection efficiency of ovarian tissue was detected by RT-PCR.Body weight,fasting blood glucose（FPG）,and fasting insulin（FSI）were measured,and HOMA-IR were calculated.HE staining was used to detect and compare the morphology of ovarian tissues in each group.Western blot was applied to detect and compare the expression of IGF2,LC3-II/I,ATG5,ATG7 and P62 proteins in ovarian tissues in each group.LC3B and P62 levels in rat ovary were detected by immunohistochemistry.Results:1.The expression level of RP11-705C15.3 was decreased in GCs of PCOS patients（P<0.0001）.In vitro experiments showed that the overexpression of RP11-705C15.3 inhibited autophagy in KGN and COV434 cells,with reduced expressions of LC3-II/I,ATG7 and ATG5,and increased expression of P62,and significantly reduced autophagosomes.2.mi R-34c-5p was significantly up-regulated in GCs of PCOS patients,and luciferase reporter as well as RNA pull-down assays confirmed its binding sites with RP11-705C15.3.RP11-705C15.3 overexpression reduced the transcription level of mi R-34c-5p in KGN and COV434 cells.The transfection of mi R-34c-5p inhibitor decreased the expression of LC3-II/I,ATG5 and ATG7,increased the expression of P62,and significantly inhibited the formation of autophagosomes in KGN and COV434 cells.The 3’UTR of IGF2 contains the binding sequence of mi R-34c-5p.Luciferase reporter assay showed that mi R-34c-5p targeted the 3’UTR of IGF2 in GCs.And IGF2 silencing enhanced autophagy in KGN and COV434 cells.3.RP11-705C15.3 enhances IGF2 expression by sponging mi R‐34c‐5p in GC-like tumor cells.The attenuated autophagy by RP11-705C15.3 over-expression was rescued by transfection with mi R-34c-5p mimics or IGF2 silencing in KGN and COV434 cells.The RP11-705C15.3/mi R-34c-5p/IGF2 axis regulates autophagy through the PI3K/Akt signal pathway.The treatment of injecting RP11-705C15.3 plasmids to PCOS rats restored the morphological characteristics in ovarian tissues of PCOS rats,as well as reduced the ovarian autophagy marked by decreased LC3-II/I and increased P62 expression.At the same time,the RP11-705C15.3 treatment partially reduced the expression of mi R-34c-5p,and enhanced the expression of IGF2 in ovarian tissues of PCOS rats.RP11-705C15.3treatment can also partially ameliorates insulin resistance in PCOS rats.Conclusion:RP11-705C15.3 was downregulated in GCs of PCOS patients as well as ovarian tissues of PCOS rats,and RP11-705C15.3 treatment alleviated the pathological changes in ovarian tissues of PCOS rats.RP11-705C15.3 enhance IGF2 expression by sponging mi R-34-5p to inhibit GCs autophagy,and partially alleviate PCOS symptoms.Therefore,RP11-705C15.3/mi R-34c-5p/IGF2 axis may play an important role in the progression of PCOS.