The Function Of MiRNAs In The Regulation Of Offspring Ovarian Granulosa Cell Apoptosis Induced By Cadmium During Pregnancy

Objective To investigate whether mi RNAs is involved in the regulation of ovarian granulosa cell apoptosis induced by cadmium during pregnancy and its mechanism,and provide a scientific basis for the epigenetic mechanism of ovarian granulosa cell damage induced by cadmium.Method Part I The Study on Screening of Apoptosis-related MiRNAs in Ovarian Granulosa Cells of Adult F1 Rats Exposed to Cadmium during Pregnancy.SPF adult SD rats were exposed to 0,0.5,2.0,and 8.0 mg/kg Cd Cl2 during pregnancy,and were intragastrically infused once a day.F1 generation females were raised to adulthood and took ovarian granulosa cells on estrus.And cells were collected for use after stable culture.The mi RNA expression profiles of the 0 and 8.0 mg/kg groups were detected using microarray chip technology.Micro RNAs related to apoptosis of ovarian granulosa cells induced by cadmium exposure during pregnancy were synthetically screened,through mi RNA chip,databases and literature.Target genes were predicted using the database for selected mi RNAs and performed bioinformatics analysis including GO and Pathway analysis.QRT-PCR was used to detect gene expression of mi RNAs in adult F1 rat granulosa cells.Part Ⅱ The Study on the Effects of MiR-16、MiR-181 b and MiR-92 a on Cadmium Induced Apoptosis of Human Ovarian Granulosa Tumor Cells(COV434).The human ovarian granulosa tumor cells was used as a tool cells.Construct COV434-tudmi R-16,COV434-tudmi R-181 b,and COV434-tudmi R-92 a cell lines mediated by lentiviral vector.The cells were divided into six groups: COV434,COV434+Cd,COV434-C+Cd,COV434-tudmi R-16+Cd,COV434-tudmi R-181b+Cd and COV434-tudmi R-92a+Cd,five groups exposed to 20 μM Cd Cl2 for 12 hours.After the exposure,collect cells.The expression of Bcl2 m RNA was detected by q RT-PCR.Western blot was used to detect the expression of BCL2.The apoptosis of COV434 in each group was detected by flow cytometry and Hoechst 33258 staining.Luciferase experiment was to verify whether mi R-92 a can be directly combined with Bcl2 3’ UTR.Part Ⅲ The Study on Effect of Cadmium Exposure during Pregnancy of F0 Rats on Apoptosis of Ovarian Granulosa Cells in Adult F2 Rats.On the basis of the F1 generation females,we chose 0 and 8.0 mg/kg groups to continue to gain F2 generation females.F2 generation females were raised to adulthood and took ovarian granulosa cells on estrus.And cells were collected for use after stable culture.The apoptosis rate of ovarian granulosa cells was detected by double staining flow cytometry.Transmission electron microscopy was used to observe the ultrastructure changes of F2 rat ovarian granulosa cells.QRT-PCR were used to detect Bcl2,Bax,Caspase3 and Caspase8 m RNA expression level.And western blot were used to detect BCL2,BAX,CASPASE3 and CASPASE8 protein expression level.QRT-PCR also was used to detect gene expression of mi R-16-5p,mi R-181b-5p and mi R-92a-2-5p in adult F2 rat granulosa cells.Detect the m RNA expression of Wt-1 m RNA in F2 rat ovarian granulosa cells.Results Part I The Study on Screening of Apoptosis-related MiRNAs in Ovarian Granulosa Cells of Adult F1 Rats Exposed to Cadmium during Pregnancy.In mi RNA ship,Fold Change is more than 1.5 or less than or equal to 0.67 and p<0.05 is the threshold for screening significant differentially expressed mi RNA were obtained 7 mi RNAs.Then,FC is more than 1.7 as threshold screened differentially expressed mi RNAs,which were 75 mi RNAs.Totally,82 mi RNAs were as tips for mi RNA chip.Target mi RNAs were predicted for Bcl2,Bcl2l1 and Bcl2l2 apoptotic genes.And target mi RNAs with SUM≥2 were included as candidates.Finally,mi RNAs that are associated with apoptosis and are more studied wered as candidates.Combined with the above,31 mi RNAs that may be related to apoptosis of ovarian granulosa cells in adult F1 rats induced by cadmium exposure during pregnancy were screened out.GO analysis showed that the target genes of these mi RNAs were mainly about cytoplasm,nucleus and cell membrane;related to protein binding,transcriptional activity,DNA binding of molecular function;biological function in the process of the main classification in cell apoptosis,transcriptional regulation,cell proliferation and so on.Pathway analysis suggested that target genes of mi RNAs might be involved in 117 pathwyas(p<0.05)including the regulation of pathway in cancer,phosphatidylinositol signaling system and so on.Compared with the control group,the changes of mi R-92a-2-5p,mi R-16-5p,mi R-181b-5p,mi R-628 and mi R-1-3p expression in were significantly increased.The difference was statistically significant(p<0.05).The trend is consistent with the results of the mi RNA chip.Part Ⅱ The Study on the Effects of MiR-16、MiR-181 b and MiR-92 a on Cadmium Induced Apoptosis of Human Ovarian Granulosa Tumor Cells(COV434).Compared with COV434,the expression of BCL2 protein in COV434 + Cd group was statistically down-regulated(p<0.05).And compared with the COV434+Cd group,the m RNA and protein expression of Bcl2 in COV434-C+Cd group were down-regulated.However,the COV434-C+Cd group didn’t lead to the up-regulation of Bcl2 expression and didn’t have a negative effect on the results of this study.Compared with the COV434+Cd group,the m RNA and protein expression of BCL2 were significantly increased.And the difference was statistically significant(p<0.05).There was no significant difference in expression of Bcl2 m RNA and protein in COV434-tudmi R-181b+Cd group(p>0.05).Compared with the COV434+Cd group,the cell apoptosis rate of COV434-C+Cd group was not statistical significance(p>0.05).But comparing with the COV434+Cd group,the number of apoptotic cells decreased significantly in the COV434-tudmi R-16 and COV434-tudmi R-92 a group(p<0.05);there was no significant difference in cell apoptosis rate of COV434-tudmi R-181b+Cd group(p>0.05).There were more strongly blue fluorescence in COV434+Cd and COV434-C+Cd group.However,the nucleus of COV434-tudmi R-16+Cd,COV434-tudmi R-181b+Cd and COV434-tudmi R-92a+Cd group is mostly blue,which with strong blue fluorescence is scattered.Compared with the Luc group,when the mi R-92 a and the wild-type Bcl2 3’ UTR region fluorescent expression plasmids were simultaneously transfected,the luciferase activity was significantly decreased(p<0.05).Part Ⅲ The Study on Effect of Cadmium Exposure during Pregnancy of F0 Rats on Apoptosis of Ovarian Granulosa Cells in Adult F2 Rats.Compared with the control group,the number of apoptotic cells and apoptotic bodies didn’t increased in high group,but mitochondria of the ovarian granulosa cells were swollen.Compared with the control group,the m RNA and protein expression of Bcl2 and Bcl2/Bax were significant up-regulated in high group(p<0.01);and protein expression of Bax were significant down-regulated in high group(p<0.01).There was significant difference in expression of Caspases3 m RNA and protein in high group(p<0.01).And there was significant difference in expression Caspases8 m RNA in high group comparing with the control group.Compared with the control group,the expression levels of mi R-16-5p and mi R-181b-5p in high group were up-regulated and the difference was statistically significant(p<0.01);while the expression level of mi R-92a-2-5p in high group was significant down-regulated(p <0.01).Compared with the control group,the expression level of Wt-1 in high group was up-regulated and the difference was statistically significant(p < 0.05).Conclusion 1.MiR-92a-2-5p,mi R-16-5p,mi R-181b-5p,mi R-628 and mi R-1-3p may promote the apoptosis of ovarian granulosa cells in F1 adult rats exposed to cadmium during pregnancy.2.Exposure to cadmium during pregnancy,mi R-16 and mi R-92 a were up-regulate and participate in the regulation of ovarian granulosa cells apoptosis in F1 adult rats through targeting Bcl2.3.There was no obvious apoptosis in ovarian granulosa cells of F2 adult rats after F0 exposed to cadmium during pregnancy.And the down-regulation of mi R-92 a may play a significant role.