Role Of MiRNA In The Hormonal Synthesis Of Ovarian Granulosa Cells In Adult F1 And F2 Rats After Cadmium Exposure During Pregnancy

Objective:Using molecular biology technology to research and analysis molecular mechanism of estrogen and progesterone synthesis dysfunction in the hormonal synthesis of ovarian granulosa cells in adult F1 rats after cadmium exposure during pregnancy.To provide a scientific basis for further research on the mechanism of female reproductive toxicity of cadmium.Method:After cage pregnancy,SPF adult SD rats were exposed to 0,0.5,2,and 8mg/kg CdCl₂ on the 1st to 20th days of pregnancy,and were intragastrically infused once a day.Took ovarian granulosa cells from F1 generation females on the 56th day after birth to stably culture for 24h and adhered to collect cells and supernatant for use.Then take the male and female of F1 generation of adult rats ratio to 2:1 cages to conceive,F2 females rats were killed on the 56th day after birth and took control group?0mg/kg?and high dose group?8mg/kg?ovarian granulosa cells to stably culture for 24h and adhered to collect cells and supernatant for use.1 Screening miRNAs related to ovarian granulocyte cytokine synthesis gene in F1generation of adult ratsTreatment of rat ovarian granulosa cells with 8mg/kg cadmium for 24 h,gene chips were prepared by extracting total RNA with Trizol simultaneously with the control cells and high dose group.Analyzed differentially expressed target genes of microRNAs in GO and pathway;analyzed of differentially expressed miRNA target genes by Miranda,mirbase,miRdb,and TargetScan prediction software and selected cadmium may affect the expression of estrogen and progesterone synthesis-related miRNAs in rat ovarian granulosa cells;tested and verified by using qRT-PCR miRNAs which selected from may affect the synthesis of estrogen and progesterone in ovarian granulosa cells of adult F1 generation cadmium exposed to cadaverine during pregnancy.2 Study on the function of miR-27a-3p and miR-10b-5p in ovarian granulosa cell2.1 Dual luciferase reporter system validates the relationship between miR-27a-3p and STAR geneConstructed STAR-3'UTR dual fluorescein reporter vector,transfected fluorescein reporter vector and cells of miRNA mimics NC,miR-27a-3p mimics,detected luciferase activity by chemiluminescence.2.2 Effects of cadmium exposure on the synthesis of estrogen and progesterone in human egg granuloma cell lines silenced by miR-27a-3p and miR-10b-5p?1?Constructed human egg granuloma cell line silenced miR-27a-3p and miR-10b-5p;?2?The experiment is divided into five groups,blank group COV434?COV434+CdCl₂?COV434-NC+CdCl₂?COV434-miR-10b-5p+CdCl₂ and COV434-miR-27a-3p+CdCl₂,removed the blank group and treated the remaining four groups with 20?mol/L cadmium chloride;?3?Determination of STAR gene expression in each group of cells by real-time PCR;?4?Western Blot assay for STAR protein expression in each group of cells?5?Determination of estradiol and progesterone levels in COV434 cells treated with CdCl₂ by Elisa assay;3 Effects of cadmium exposure on hormonal cytokines synthesis in ovarian granules of adult rats during pregnancy?1?Determination of estradiol and progesterone levels in in ovarian granulosa cells of adult F1 generation treated with CdCl₂ by Elisa assay;?2?Expression levels of estrogen and progesterone synthesis related genes STAR,CYP11a1 and SF-1 in ovarian granulosa cells of F2 generation adult rats induced by cadmium exposure during pregnancy?qRT-PCR and Western Blot?;?3?Expression levels of miR-10b-5p and miR-27a-3p in ovarian granulosa cells of F2 adult rats induced by cadmium exposure during pregnancy?qRT-PCR?Results1 Screening miRNAs related to ovarian granulocyte cytokine synthesis gene in F1generation of adult ratsThe results of Gene chip:High-dose group miRNA expression profile compared with control group,the expression of 232 miRNA had changed,111 miRNAs with increased expression?FC?1.5?,121 miRNAs with decreased expression?FC?0.67?.According to GO analysis,it was found that three biological processes related to estrogen and progesterone were enriched,they were intracellular estrogen and progesterone receptor signaling pathway,steroid hormone-mediated signaling pathway and cholesterol metabolism.According to the results of pathway found 4 pathways related to estrogen and progesterone action,they were MAPK signal transduction pathway,progesterone-mediated oocyte maturation,estrogen and progesterone signaling pathways,and PI3K-Akt signaling pathway.According to the GO and Pathway analysis based on expression profiles of miRNA microarray chips,online prediction software and literature,screened 10 miRNAs related to estrogen and progesterone synthesis and pathways.The results of qRT-PCR:Compared with the control group,8 miRNAs were differentially expressed.The expression level of miR-27a-3p was up-regulated in 0.5,2.0,and 8.0 mg/kg cadmium-containing groups,miR-10b-5p expression level up-regulated in 2.0,8.0mg/kg cadmium-containing group,both miRNA in dose-effect relationship?P<0.05?.2 Study on the function of miR-27a-3p and miR-10b-5p in ovarian granulosa cell2.1 Dual luciferase reporter system validates the relationship between miR-27a-3p and STAR gene?1?The results show that compared to the wild-type miRNA NC+STAR-3UTR,the luciferase activity of co-transfected miR-27a-3p+STAR-3UTR was significantly decreased.?2?Compared to the mutant miRNA NC+STAR-3UTR,there was no significant change in the luciferase activity of the mutant miR-27a-3p+STAR-3UTR.2.2 Effects of cadmium exposure on the synthesis of estrogen and progesterone in human egg granuloma cell lines silenced by miR-27a-3p and miR-10b-5p2.2.1 Determination of STAR gene expression in each group of cellsCompared with the COV434-NC+CdCl₂ group and the COV434+CdCl₂ group,STAR gene mRNA and protein expression levels were not statistically significant?P>0.05?.Compared with the COV434 group,the expression levels of STAR mRNA and protein were decreased in the COV434+CdCl₂ group?P<0.05?.Compared with the COV434+CdCl₂ group,the expression level of STAR mRNA and protein in the experimental group inhibitor+CdCl₂ were increased?P<0.05?.2.2.2 Determination of estradiol and progesterone levels in COV434 cells treated with CdCl₂ by Elisa assayThere was no statistically significant difference between the COV434+CdCl₂group and the COV434-NC+CdCl₂ group?P>0.05?.Decreased levels of estradiol and progesterone in the COV434+CdCl₂ group compared with the COV434 group?P<0.05?.Compared with the COV434+CdCl₂ group,the level of inhibitor+CdCl₂ estradiol and progesterone levels in the experimental group increased?P<0.05?.3 Effects of cadmium exposure on hormonal cytokines synthesis in ovarian granules of adult rats during pregnancyCompared with the control group,the progesterone secretion level decreased in the cadmium-dose group?P<0.05?and estradiol secretion levels were not statistically significant.Compared with the control group,mRNA and protein expression decreased in the cadmium-dose group of SF-1.Increased mRNA expression and decreased protein expression in cadmium-dose group STAR and CYP11a1 groups?P<0.05?.Increased expression levels of miR-27a-3p and miR-10b-5p in the cadmium-treated group compared with the control group.ConclusionBased on the above results,the following conclusions can be drawn:1.Screening 10 miRNAs related to estrogen and progesterone synthesis in ovarian granulosa cells of cadmium-exposed F1 progeny rats during pregnancy.2.MiR-27a-3p and miR-10b-5p regulate the synthesis of estrogen and progesterone in ovarian granulosa cells of adult F1 rats induced by cadmium exposure during pregnancy.3.Cadmium still affects the progesterone synthesis function of F2 generation,miR-27a-3p and miR-10b-5p affect the synthesis of estrogen and progesterone by inhibiting the translation process of STAR gene...