| [Purpose] To isolate granulosa cells derived from follicular fluid,analyze the expression of miR-484 and its correlation with assisted reproductive outcomes in patients with different ages and different ovarian function.[Methods] Follicular fluid samples of female patients undergoing assisted reproduction technology(ART)for infertility were collected at reproductive medicine center.Granulosa cells were separated by density gradient centrifugation;its FSHR positive rate was identified by immunofluorescence.All collected samples were divided into young group(≤35 years old)and aging group(>35 years old)according to age;divided into normal ovarian reserve(NOR)group and diminished ovarian reserve(DOR)group according to ovarian function.RNA was extracted from granulosa cells of each group,relative expression of miR-484 was detected by q RT-PCR.Analyze the correlation between miR-484 relative expression level and age,basal sex hormone level,ssisted reproduction outcomes.[Results] A total of 114 samples met the criteria were collected.Immunofluorescence results showed that the positive rate of FSHR of the extracted granular cells was > 90%.The relative expression level of miR-484 was significantly higher in DOR group than NOR group(P<0.0001);there was no statistical difference(P>0.05)between aging group and young group though the former was slightly higher.The relative expression level of miR-484 was significantly positively correlated with basic sex hormone FSH(r=0.2654,P<0.05),negatively correlated with AMH level(r=-0.4008,P<0.001)and numbers of AFC(r=-0.4096,P<0.001),and no obvious linear relationship with LH,Estradiol,Progesterone(P>0.05).About the assisted reproduction outcomes,the relative expression level of miR-484 was significantly negatively correlated with the numbers of oocyte retrieved(r=-0.4777,P <0.0001)and the rate of good quality embryo(r=-0.31,P<0.01).As for the rate of normal fertilization rate,there was no obvious linear relationship.(P>0.05).[Conclusions] Granulosa cells extracted by density gradient centrifugation are highly purified;the relative expression of miR-484 is significantly increased in DOR patients,and closely related to ovarian reserve and assisted reproduction outcomes.[Purpose] To determine the relative expression of miR-484 in different tissues of mice,ovarian tissues at different times,and the localization of miR-484 in main cell types of ovary and 293 T cells.[Methods] Tissues of heart,liver,spleen,lung,kidney,brain,cerebellum,hypothalamus,stomach,intestine,muscle,ovary,oviduct,uterus and testical were collected from C57BL/6J mice at 6-8 weeks;ovaries were collected at 1 day,1 week,2 weeks,3weeks,and 8 weeks after born.RNA was extracted and the relative expression of miR-484 was detected by q RT-PCR.Fluorescence in situ hybridization(FISH)was used to detect the localization of miR-484 in mouse granulosa cells,cumulus cells,GV oocytes and commonly used 293 T cells.[Results] miR-484 was detected in heart,liver,spleen,lung,kidney,brain,cerebellum,hypothalamus,stomach,intestine,muscle,ovary,fallopian tube,uterus and testical.The relative expression of miR-484 was lowest in testical,highest in cerebellum,and about 7times in ovary than testical.After born,the expression level of miR-484 in mice ovary gradually increased,reaching about the same level at 3 weeks as at 8 weeks.miR-484 was expressed both cytoplasm and nucleus in mouse granulosa cells,cumulus cells,293 T cells and almostly nucleus in GV oocytes.[Conclusions] miR-484 is widely expressed in various tissues of C57BL/6J mice at6-8 weeks and has different cell localization in different cell types.[Purpose] To identify the selected granulosa cell line COV434,determine the optimal concentration and time of miR-484 mimic/inhibitor transfection,and define whether miR-484 over expression/inhibition can affect the function of granulosa cells by regulating the division and fusion of mitochondria.[Methods] Immunofluorescence and FISH were used to identify the positive FSHR rate and expression pattern of miR-484 in COV434.30 n M,50 n M,and 100 n M Cy3-labeled mimic-NC were transfected into COV434,the optimal transfection concentration was determined 24 hours later according to the immunofluorescence intensity.Transfect miR-484 mimic to COV434 with the optimal concentration;collect cells at 24 hours,48 hours,and 72 hours after transfection.The relative expression of miR-484 was determined by q RT-PCR to determine the optimal transfection time.After transfected miR-484 mimic and inhibitor,the relative expression of mitochondrial division and fusion genes(MFN1、MFN2、OPA1、DRP1、FIS1)m RNA were determined by q RT-PCR;ATP levels of granulosa cells were detected by microplate reader and CCK-8 was used to detect granulosa cells proliferation activity.[Results] Immunofluorescence results showed that the positive rate of FSHR in COV434 was>90%,and COV434 had the same miR-484 expression pattern as granulosa cells in mice: both cytoplasm and nucleus were expressed.Fluorescence intensity after transfection of mimic-NC labeled with Cy-3 at different concentrations showed the highest transfection efficiency at 100 n M.After transfection at a concentration of 100 n M,miR-484 relative expression all increased at three time points.It is most significant at 24 h,and then gradually decreased with time.Therefore,100 n M and 24 h was used as the optimal transfection concentration and detection time point after transfection.Compared with mimic-NC group,mitochondrial fusion genes MFN1,OPA1 m RNA relative expression in miR-484 mimic transfection group was significantly reduced(P<0.05;P<0.01),MFN2 m RNA relative expression also decreased,but no statistics difference(P> 0.05);the relative expression levels of mitochondrial division genes DRP1 and FIS1 m RNA were significantly increased(P<0.01;P<0.01).Compared with inhibitor NC group,mitochondrial fusion genes MFN1,OPA1 m RNA relative expression in miR-484 inhibitor transfection group was significantly increased(P<0.05;P<0.001),relative expression of MFN2 m RNA increased too,but no statistics difference(P>0.05);the relative expression levels of mitochondrial division genes DRP1 and FIS1 m RNA all showed a downward trend,and there was still no statistical difference(P>0.05).Compared with mimic-NC group,the proliferation activity and mitochondrial ATP level in miR-484 mimic transfection group decreased significantly at 24h(P<0.01;P<0.05)and 48h(P<0.05;P<0.05)after transfection.Compared with inhibitor NC group,the proliferation activity and mitochondrial ATP level in miR-484 inhibitor transfection group increased significantly at 24 h after transfection(P<0.01;P<0.05),but no statistical difference at 48h(P> 0.05;P> 0.05),though they showed an increasing trend.[Conclusions] miR-484 can affect the function of granulosa cells and mitochondrial function by regulating mitochondrial division and fusion. |
PDF Full Text Request