Mechanism of lncRNA GNAQ-AS1 in high androgen polycystic ovary syndrome rats
Objective To investigate the role and mechanism of long non-coding RNA GNAQ-AS1-mediated miRNA-20b-5p targeting cell division cycle and apoptosis regulatory protein 2(CDCD2)in high androgen polycystic ovary syndrome(PCOS)rats.Methods At the cellular level,the optimal inhibitory concentrations of LncRNA GNAQ-ASl and miRNA-20b-5p were detected by cell counting kit-8(CCK-8),and the migration of cells was detected by Transwell chambers.The cell cycle was detected by flow cytometry.At the animal level,rats were randomly divided into four groups:control group,model group,lncRNA GNAQ-AS1 siRNA group and miRNA-20b-5p mimics group,with 10 rats in each group.The model group was continuously administered with letrozole dissolved in 1％carboxymethyl cellulose at a dose of 1 mg·kg-1·d-1 for 3 weeks,3 times a week.The lncRNA GNAQ-AS1 siRNA group was treated with siRNA to silence the expression of lncRNA GNAQ-AS1 on the basis of establishing the model,with injection once a week for 3 weeks.The miRNA-20b-5p mimics group was treated with overexpression of miRNA-20b-5p by miRNA-20b-5p mimics,administered via the tail vein,with the dose of 1 mg·kg-1,injected once a week for 3 weeks.The control group was injected with an equal volume of 0.9％NaCl.After the experiment,the rats were sacrificed and blood samples,ovarian tissue RNA and protein were extracted.The serum luteinizing hormone(LH),testosterone(T),serum anti-Mullerian hormone(AMH),and follicle-stimulating hormone(FSH)expression levels were detected by enzyme-linked immunosorbent assay(ELISA)kit.The expression of lncRNA GNAQ-AS1,miRNA-20b-5p and CDCD2 mRNA in ovarian tissue was detected by real-time quantitative polymerase chain reaction(RT-qPCR)technology,and the expression level of CDCD2 was detected by Western blot.Results The results of the CCK-8 assay showed that when 5 μmol·L-1lncRNA GNAQ-AS1 siRNA was administered,the cell viability of the control group and lncRNA GNAQ-AS1 siRNA group were(38.25±0.62)％and(86.14±3.13)％.When 10 μmol·L-1 miRNA-20b-5p mimics were administered,the cell proliferation inhibition rate of control group and miRNA-20b-5p mimics group were(40.70±0.32)％and(85.35±2.13)％,indicating that KGN cell viability significantly decreased when 10 μmol·L-1miRNA-20b-5p mimics were administered.At the animal level,RT-qPCR results showed that the expression levels of lncRNA GNAQ-AS1,miRNA-20b-5p,and CDCD2 mRNA in model group were 1.72±0.37,0.59±0.10 and 2.47±0.13,respectively,indicating that the expression levels of lncRNA GNAQ-AS1 and CDCD2 mRNA were significantly up-regulated in model group,while the expression level of miRNA-20b-5p was down-regulated compared with control group(P＜0.05).Compared with control group,the levels of LH,T and AMH in serum were significantly increased in model group(all P＜0.05),while the levels of FSH and E2 were significantly decreased(P＜0.05).Compared with model group,lncRNA GNAQ-AS1 siRNA and miRNA-20b-5p mimics groups significantly reduced the levels of LH,T and AMH in serum and increased the levels of FSH and E2.In addition,the protein expression level of CDCD2 was significantly increased in both lncRNA GNAQ-AS1 siRNA and miRNA-20b-5p mimics groups.Conclusion LncRNA GNAQ-AS1 mediates the targeted regulation of miRNA-20b-5p to play an important role in CDCD2 in PCOS.
long non-coding RNAguanine nucleotide binding protein q polypeptide-antisense RNA1polycystic ovarian syndromemolecular mechanism
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