| Objective: MiRNAs play an important role in ovarian physiology and pathology.Some studies have shown that miRNAs play an important role in the occurrence of ovarian cancer,activation of primordial follicles,follicular development,oocyte maturation and ovulation.Therefore,in this study,explored the level differences of miR-23 a in serum of polycystic ovary syndrome(PCOS)patients and healthy women,and the effects of miR-23 aexpression levels on the biological behaviors such as ovarian granulosa cell proliferation and apoptosis.The aim is to provide limited theoretical support and experimental data for the application of miRNAs to the diagnosis and treatment of pcos.Chapter 1 miR-23 a levels in serum of PCOS patients and healthy volunteerMethods: The serums of 50 Chinese Han women(30.98 ± 3.82 years old)who were diagnosed with PCOS and50 healthy Han women(30.76 ± 3.12 years old)matched with age at the same time were collected in the Fujian Ningde Mingong Hospital from September 2018 to December 2018.Body mass index(BMI)was calculated.Sex hormones levles were assessed by radioimmunoassay.The concentration of miR-23 a in serum was detected by qRT-PCR.In addition,the association between serum levels of miR-23 a and BMI and sex hormone levels was analyzed.Results: The serum level of miR-23 a was obviously lower in PCOS patients compared to healthy volunteer(P< 0.001).There was a positive correlation between serum miR-23 a levels and BMI in PCOS patients(P = 0.0199,r = 0.3285),but this correlation was not found in healthy controls(P = 0.8632,r = 0.02499).The concentration of luteinizing hormone(LH)in serum of PCOS patients were 9.35 ± 1.77 mIU/mL.There was a negative correlation between serum miR-23 a concentration and LH concentration in PCOS patients(P = 0.0088,r =-0.3665),but this correlation was not found in healthy controls(P = 0.3210,r = 0.1432).Conclusion: The serum concentrations of miR-23 a were lower in PCOS patients compared to healthy volunteer.In addition,we confirmed the positive effects of obesity and LH levels on circulating miR-23 a levels.Chapter 2 MiR-23 a expression is associated with proliferation and apoptosis of human ovarian granulosa cells.Methods: The expression of miR-23 a in human ovarian granulosa cells cov434 was overexpressed or knocked down by RNA interference technique.The effects of miR-23 a on cell proliferation,cell cycle and apoptosis were determinated by CCK8 assay and flow cytometry,respectively.Results: Transfection of miR-23 a mimic NC and inhibitor NC did not cause significant changes in cell proliferation compared to normal cultured cells(control group).Transfection of miR-23 a mimic inhibited cov434 cells proliferation(P< 0.05).In contrast,transfection of miR-23 a inhibitor promoted cov434 cells proliferation(P< 0.05).Transfection of miR-23 a mimic later,cell cycle was arrested in the G0/G1 phase,and proportion of cells in the S phase and G2/M phase was decreased(P< 0.05).In contrast,transfection of miR-23 a inhibitor later,the proportion of G2/M phase cells was increased(P< 0.05),while the proportion of G0/G1 phase and S phase cells was decreased(P< 0.05).Moreover,miR-23 a mimic transfectionsignificantly increased the proportion of apoptotic cells in early and late stages(P< 0.05);Transfection of miR-23 a inhibitor decreased proportion of apoptotic cells(P< 0.05).Conclusion:MiR-23 a expression is assocaited with proliferation and apoptosis in human ovarian granulosa cells.Overexpression of miR-23 a inhibits proliferation by arresting cell cycle and promotes apoptosis.Chapter 3 FGD4 is the new downstream target of miR-23aMethods:The bioinformatics software Target Scan screened out the potential miR-23 a binding site of FGD4,,and the dual luciferase reporter assay verified the direct binding of the two.The effects of miR-23 a expression on the FGD4 expression were detected.Results: Co-transfection of miR-23 a mimic inhibited the luciferase activity of the FGD4-WT plasmids(P< 0.01),but did not inhibit the FGD4-Mut plasmid luciferase activity.This result indicates that miR-23 a and FGD4 mRNA do bind directly through the predicted site.Transfection of miR-23 a mimic reduced intracellular FGD4 expression(P< 0.01);in contrast,Transfection of miR-23 a inhibitor increased intracellular FGD4 expression(P< 0.05).In addition,transfection of miR-23 a mimic significantly increased intracellular expression of CDC42 and PAK-1(P< 0.01);in contrast,transfection of miR-23 a inhibitor significantly reduced the expression of actived CDC42(GTP bround)and p-PAK-1 in cells(P< 0.05).Conclusion: In this study,we found a new target for miR-23 a in human ovarian granulosa cells: FGD4. |
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