| Objective: Polycystic ovarian syndrome(PCOS)is characterized by chronic anovulation,insulin resistance and hyperandrogenemia.Its etiology is still not yet clear.The potential pathogenic factors include genetic factors,environmental factors,lifestyle factors and so on.In the early stage of follicular development,ovarian granulosa cells in PCOS patients proliferated excessively,and its apoptosis rate was low,which led to a large number of preantral follicles recruited at the same time and began to develop.Moreover,PCOS patients have delayed growth of antral follicles,longer proliferation period,and delayed atresia,resulting in most follicles stagnating at a certain growth stage and accumulating.Excessive proliferation and decreased apoptosis of granulosa cells will lead to dysfunction of granulosa cells in the early stage of follicular development,and then participate in the occurrence and development of PCOS.Long chain noncoding RNA(lnc RNAs)refers to RNA whose transcripts are between 200-100000 nucleotides in length and do not have the ability of protein translation.It has been reported that a variety of lnc RNAs can participate in PCOS by affecting chromatin remodeling,transcriptional control,post transcriptional processing and so on.Lnc-FRMD6-1:1 is one of lncrnas.Up to now,the function of lnc-FRMD6-1:1 has not been reported,and its role in PCOS is not clear.We previously found that the expression of lnc-FRMD6-1:1 in PCOS patients was higher than that in normal control group in the follicular fluid.Therefore,we designed this experiment to confirm the role of lnc-FRMD6-1:1 in PCOS and its mechanism.Under normal circumstances,period changes of ovarian are regulated by gonadotropins and growth factors.Insulin like growth factor-1(IGF-1)is over activated in PCOS,and then acts on IGF-1R and insulin receptor,causing a series of reactions.But little attention has been paid to the role of IGF-1R in this process.miR-330-3p is a kind of miRNA.miRNA is transcribed by RNA polymerase in the nucleus,then processed into miRNA precursor with stem ring structure,transported to the cytoplasm,and cut into mature miRNA,participating in many biological functions.In this study,we verified the role of lnc-FRMD6-1:1 in PCOS through miR-330-3p and IGF-1R.Methods: 1.By summarizing the sequencing results reported in the published articles and bioinformatics analysis of the online database,the possible differential genes between PCOS patients and normal controls were screened,and their regulatory relationships and possible binding sites were predicted.Collect ovarian granulosa cell samples from PCOS patients and normal controls,and test the expression of lnc-FRMD6-1:1,miR-330-3p and IGF-1R in ovarian granulosa cells of polycystic ovary syndrome by q PCR.The PCOS mouse model was established by DHEA+HFD.The expression of lnc-FRMD6-1:1,miR-330-3p and IGF-1R in the ovaries of PCOS mice and normal control mice were detected.2.RNA22 tool was used to predict the binding sites between miR-330-3p and lnc-FRMD6-1:1 and the 3 ’UTR region of IGF-1R.Lnc-FRMD6-1:1 si RNA and lnc-FRMD6-1:1 overexpression plasmid were transfected into human ovarian granulosa tumor cell lines KGN and COV434 to construct lnc-FRMD6-1:1 knockdown and overexpression cell models,which were verified by q PCR.The expression of miR-330-3p was also detected by q PCR.KGN and COV434 cells were transfected with miR-330-3p mimics and inhibitor to construct miR-330-3p knockdown and overexpression cell models,which were verified by q PCR.The expression of lnc-FRMD6-1:1 and IGF-1R were also detected by q PCR.WB was used to detect the expression of IGF-1R at the protein level.The expression of IGF-1R in cells was further verified by immunofluorescence.Lnc-FRMD6-1:1 and IGF-1R mutant and wild-type plasmids were used to co transfect tool cells with miR-330-3p mimics and mimics NC,respectively.Verify the binding sites between them.3.Lnc-FRMD6-1:1knockdown and overexpression cell models and miR-330-3p knockdown cell models were constructed,and their effects on cell proliferation were detected by CCK-8experiment.MTT assay was used to detect its effect on cell proliferation.Flow cytometry was used to detect its effect on apoptosis,and WB was used to detect apoptosis related proteins.Lnc-FRMD6-1:1 si RNA,lnc-FRMD6-1:1 si RNA+miR-330-3p inhibitor and Lnc-FRMD6-1:1 si RNA nc+miR-330-3p inhibitor NC was transfected into KGN and COV434 cells respectively.The effects on cell proliferation and apoptosis were detected by the above experiments.It was proved that miR-330-3p inhibitor partially responded to the changes in cell behavior caused by lnc-FRMD6-1:1 knockdown.Finally,the PCOS mouse model was established and randomly divided into two groups.One group was injected with miR-330-3p agomir via caudal vein,and the other group was injected with miR-330-3p agomir NC for three days.Fasting blood glucose was collected three days after the injection,body weight was recorded and ovarian size was observed.PCOS recovery was observed by HE staining.The effect of agomir on lnc-FRMD6-1:1 and IGF-1R was detected by q PCR.Results: 1.Compared with the control group,lnc-FRMD6-1:1 and IGF-1R were highly expressed in ovarian granulosa cells of PCOS patients,while miR-330-3p was low expressed.The estrous cycle of mice in PCOS group was disordered and ovulated irregularly.Compared with the control group,lnc-FRMD6-1:1 and IGF-1R in the ovaries of mice in PCOS group showed high expression,while miR-330-3p showed low expression.2.The predicted results showed that there were possible binding sites between miR-330-3p and lnc-FRMD6-1:1 and IGF-1R 3 ’UTR region.q PCR showed that miR-330-3p was significantly up-regulated in the cell model with lnc-FRMD6-1:1knockdown,but down regulated in the cell model with lnc-FRMD6-1:1 overexpression.The expression of lnc-FRMD6-1:1 and IGF-1R decreased at RNA level after transfection of miR-330-3p mimics.WB and immunofluorescence experiments showed that the expression of IGF-1R decreased at protein level after transfection of miR-330-3p mimics.The expression of lnc-FRMD6-1:1 and IGF-1R increased at RNA level after transfection of miR-330-3p inhibitor.WB and immunofluorescence experiments showed that the expression of IGF-1R was up-regulated at protein level after transfection of miR-330-3p inhibitor.The results of double luciferase reporter gene showed that the relative luciferase activity of wild-type lnc-FRMD6-1:1+miR-330-3p mimics and wild-type igf-1r+miR-330-3p mimics was lower than that of the other three groups,which verified that there might be binding sites between miR-330-3p and lncrna lnc-FRMD6-1:1 and IGF-1R.3.CCK-8 test,MTT test,annexin V-FITC apoptosis detection and WB detection of apoptosis related proteins showed that after lnc-FRMD6-1:1 knockdown,cell proliferation decreased and apoptosis increased.After overexpression of lnc-FRMD6-1:1,cell proliferation increased and apoptosis decreased.After knockdown of miR-330-3p,cell proliferation increased and apoptosis decreased.miR-330-3p inhibitor partially restored the changes in cell proliferation and apoptosis caused by lnc-FRMD6-1:1knockdown.After tail vein injection of miR-330-3p agomir,the fasting blood glucose of PCOS mice decreased slightly.Compared with agomir NC group,the ovaries of miR-330-3p agomir group were smaller,with a more rounded appearance and fewer bulges.He staining showed that the number of ovarian follicles in agomir NC group increased,some of them showed polycystic changes,no obvious corpus luteum,and the follicular cavity was huge.However,in the miR-330-3p agomir group,there were fewer follicles at various stages in the ovary,with obvious corpus luteum,and some follicles showed polycystic changes.It indicates that the PCOS symptoms are relieved.Conclusion: Compared with the control group,lnc-FRMD6-1:1 and IGF-1R were highly expressed in PCOS ovaries,while miR-330-3p was low expressed.The highly expressed lnc-FRMD6-1:1 regulates the expression of IGF-1R by competitively binding miR-330-3p,and then affects the progress of PCOS by promoting the proliferation of granulosa cells and inhibiting the apoptosis of granulosa cells. |
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