RESUMO
Cadmium (Cd) can induce ovarian injury by microRNAs (miRNAs), however, the molecular mechanism of miRNAs after Cd exposure have not known. In this study, 56-day-old adult female Sprague-Dawley (SD) rats were injection with PMSG, after 48 h, ovarian granulosa cells (GCs) were extracted and cultured for 24 h, then treated with 0, 2.5, 5, 10 and 20 µM Cd for 24 h. The results showed that expression levels of miR-92a-2-5p (upregulated) and Bcl2 (downregulated) changed significantly after Cd exposure. The messenger RNA (mRNA) and protein expression levels of DNMT1, DNMT3A, and DNMT3B had changed, but no obvious differences were found in miR-92a-2-5p single site methylation. The transcription factors C-MYC (upregulated), E2F1 (downregulated), and SP1 (downregulated), which target miRNAs significantly changed after exposure to Cd. The human ovarian GC tumor line (COV434) was used to knocked down C-myc, and the expression of miR-92a-2-5p was downregulated in the COV434-C-myc + 10 µM Cd group compared with COV434 cells. The N6-methyladenosine (m6A) methylation modification levels of long noncoding RNA (lncRNA) MT1JP and lncRNA CDKN2B-AS, which regulate miR-92a-2-5p were detected. In the 10 µM Cd group, m6A methylation levels at MT1JP-84, CDKN2B-AS-257, and CDKN2B-AS-329 were reduced. In summary, after Cd exposure, expression of miR-92a-2-5p, which targets the antiapoptotic gene Bcl2, was upregulated, which may be primarily related to upregulation of C-myc. MiR-92a-2-5p promoter DNA methylation may has no obvious effect on miR-92a-2-5p. Otherwise, the role of m6A methylation modified lncRNA MT1JP and lncRNA CDKN2B-AS in the regulation of miR-92a-2-5p needs further study.
Assuntos
Cloreto de Cádmio/toxicidade , Células da Granulosa/efeitos dos fármacos , MicroRNAs/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Transcrição Gênica/efeitos dos fármacos , Ativação Transcricional/efeitos dos fármacos , Adenina/análogos & derivados , Adenina/metabolismo , Animais , Linhagem Celular , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA , Feminino , Células da Granulosa/metabolismo , Proteínas de Choque Térmico HSP40/genética , Proteínas de Choque Térmico HSP40/metabolismo , MicroRNAs/genética , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-myc/genética , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Ratos Sprague-Dawley , Regulação para CimaRESUMO
Cadmium (Cd) is known to affect ovarian granulosa cells (GCs), but no information on the transgenerational effects of Cd on GCs. In this study, pregnant Sprague-Dawley (SD) rats were orally dosed with Cd from gestation day 1 until birth. F1 or F2 female rats were mated with untreated males to produce the F2 or F3 generation. In the F1 generation, apoptotic cell bodies were observed in the Cd-treated group but not in the F2 generation. Moreover, significant changes in B-cell lymphoma 2 (Bcl2) expression were observed in both generations. Additionally, the expression of microRNAs (miRNAs) was significantly changed based on microarray analysis. Specifically, miR-16-5p and miR-181b-5p were upregulated in F1 and F2 rats, while miR-92a-2-5p demonstrated different expression patterns between the two generations. In F3 generation, miR-16-5p and miR-92a-2-5p were down-regulated. Further, another experiment was used to show that miR-16-5p and miR-92a-2-5p regulated the Bcl2-induced apoptotic effect of Cd on GCs by the Human ovarian GC tumor line (COV434 cell line) miRNA-knockdown model Overall, the results indicate that prenatal Cd exposure has epigenetic transgenerational effect on GCs, Moreover, the underlying mechanism may involve interference with miR-16-5p and miR-92a-2-5p-mediated regulation of Bcl2 genes in offspring.