Cadmium Is Involved In Its Damage To Ovarian Cells By Anti-mullerian Negative Regulatory Stem Cell Factor

Objective To investigate whether the anti-Mullerian hormone(AMH)is negative regulatory stem cell factor(SCF)involved in cadmium-induced ovarian cell damage.And to explore the possible miRNAs which involved in the regulation of the changes in AMH expression induced by cadmium exposure.And provide a scientific basis for further elucidating cadmium toxicity and mechanism of cadmium.Methods1 The ovarian granulosa cells was exacted from 21 days old female SD rats which was cultured for 24 hours,Then the ovarian granulosa cells was culture with5 μmol/L,10 μmol/L and 20 μmol/L Cd Cl2 and the negative control group(culture medium without Cd Cl2)for 12 hours.The total RNA and total protein was extracted,and the changes of mRNA and protein expression of AMH and SCF were detected by RT-PCR and Western Blot,respectively.2 The human ovarian granulosa tumor cells was used as a tool cells,in plasmid vector delivery of AMH-sh RNA into cells in stable knockdown of AMH gene,then the experimental set up of 20 μM cadmium chloride treatment group COV434;20μM cadmium chloride in AMH-sh RNAc group;20 μM cadmium chloride in AMH-sh RNA transfection group for 12 hours.Then the total RNA and total protein was extracted,and the changes of mRNA and protein expression of AMH and SCF were detected by RT-PCR and Western Blot,respectively.3 First,miRNAs,target Scan and other databases were used to screen the miRNAs that could regulate AMH expression in combination with the literature and our mircro RNA microarray.Then,the ovarian granulosa cells was exacted from21 days old female SD rats which was cultured for 24 hours,Then the ovarian granulosa cells was culture with 5 μmol/L,10 μmol/L and 20 μmol/L Cd Cl2 and the negative control group(culture medium without Cd Cl2)for 12 hours.Then the total RNA was extracted,and the changes of the changes of miRNA related to the regulation of AMH were detected by RT-PCR,respectively.Results1 The expression of SCF mRNA in the ovarian granulosa cells exposed to cadmium exposure at 21 days of age was significantly lower than that in the control group(P <0.05).The trend test showed that the level of exposure to cadmium was dose-effect relationship with the expression level of SCF mRNA(P <0.05).The expression of SCF protein was down-regulated in the exposed group compared with the control group(P <0.05).The trend test showed that the level of exposure to cadmium was dose-effect relationship with the expression level of SCF protein(P <0.05).At the same time,the expression level of AMH mRNA was increased in the exposed group compared with the control group(P <0.05).The trend test showed that the level of exposure to cadmium was dose-effect relationship with the expression level of AMH mRNA(P < 0.05).Compared with the control group,the expression level of AMH protein increased,and the expression level of AMH protein in 20 μmol/L Cd Cl2 group was significantly higher than that in control group(P <0.05).Trend test showed that the level of exposure to cadmium and AMH protein expression was dose-dependent relationship(P <0.05).2(1)The transfection efficiency was optimized by GFP-labeled negative control to obtain cell transfection efficiency after 24 hours.Suggesting that the survival of the cov434 cells were successfully transfected with botulinumone,the transfection efficiency was 100%.COV434 cells were transfected with AMH-sh RNA and AMH-C,respectively.Results AMH-sh RNA was transfected into AMH-C in control group compared with AMH-C in control group.Compared with AMH-C in AMH-sh RNA group,AMH mRNA and AMH protein expression levels were significantly decreased,AMH mRNA and AMH protein decreased by more than 70%.(2)20 μmol/L Cd Cl2 exposure AMH gene knockdown group(cov434-AMH-sh RNA)as compared to the control group(COV434 cell)exposed to 20 μmol/L Cd Cl2,reduced the mRNA expression level of AMH while SCF mRNA expression levels increased,the difference was statistically significant(P<0.05).20 μmol/L Cd Cl2 exposure vector control group(cov434-AMH-Control)as compared to the control group(COV434 cell)exposed to 20 μmol/L Cd Cl2,AMH mRNA expression levels did not change significantly statistical differences SCF mRNA expression levels(P <0.05).20 μmol/L Cd Cl2 AMH gene knockdown Infected group(cov434-AMH-sh RNA)group as compared to 20 μmol/L Cd Cl2 vector control group(cov434-AMH-Control)the mRNA expression levels decreased AMH while SCF mRNA expression increased(P <0.05).The difference was statistically significant(P <0.05).(3)Compared with 20 μmol/L Cd Cl2-treated cov434 cells,the expression level of AMH protein was significantly decreased while the expression of SCF protein was increased,but the expression of AMH protein was different(P <0.05).There was no significant difference in SCF protein expression between the two groups(P> 0.05).The expression of AMH protein was significantly lower in the vctor control group(cov434-AMH-Control)compared with 20 μmol/L Cd Cl2-treated cov434 cells treated with 20 μmol/L Cd Cl2(P <0.05),and the difference was statistically significant(P <0.05),But there was no significant difference in the expression level of SCF protein,the difference was not statistically significant(P> 0.05).The expression of AMH protein was significantly lower in the AMH gene knockdown group(cov434-AMH-sh RNA)than exposed to20 μmol/L Cd Cl2 in the vector control group with 20 μmol/L Cd Cl2(cov434-AMH-Control)while the SCF protein(P <0.05).There was no significant difference in the expression of SCF protein between the two groups(P> 0.05),but there was no significant difference in the expression of AMF protein(P> 0.05).3 The expression levels of miR-129-5p,miR-486,miR-503-3p and miR-871-3p were decreased in the ovarian granulosa cells exposed to cadmium,and miR-133a-5p,miR-138-5p,miR-325-3p,miR-615 expression levels increased.Conclusions1 Cadmium is involved in ovarian cell injury in rats by Anti-Müllerian hormone negatively regulates the expression of stem cell factor.2 Mi R-129-5p,miR-486,miR-503-3p,and miR-871-3p may be involved in the regulation of AMH expression induced by cadmium exposure in the granulosa cells of SD rats exposed to cadmium..