Effect of cadmium on kitl pre‐mRNA alternative splicing in murine ovarian granulosa cells and its associated regulation by miRNAs

Abstract

In this study, we established an in vitro exposure model of murine ovarian granulosa cells to observe the effect of Cd on alternative splicing of the kitl pre-mRNA and subsequently to explore the role of kitl gene expression regulation-related miRNAs through miRNA prediction, miRNA chip, bioinformatics and real-time quantitative polymerase chain reaction analyses. Our results showed that the kitl1/kitl2 mRNA ratio was significantly different (P < 0.05) at different dosages and times. The miRNA chip analysis showed that the miRNA expression profiles for the Cd treatment were significantly changed, and the expression of 29 miRNAs involved in alternative splicing of the kitl pre-mRNA was changed. The gene ontology analysis showed that the target gene functions of these 29 miRNAs were mainly enriched in the biological processes of cell metabolism regulation, post-transcriptional regulation of mRNA, interleukin-6-mediated signal transduction, cell cycle, cell proliferation, differentiation and migration. The pathway enrichment analysis showed that the target genes of the differentially expressed miRNAs were mainly enriched in the Ras signaling pathway, the Rap1 signaling pathway, the Foxo signaling pathway, the Hippo signaling pathway, the MAPK signaling pathway and the carcinogenic pathway. Polymerase chain reaction verification results showed that compared to the control group, the variation trends in the expression of mmu-miR-27a-3p, mmu-miR-34b-5p, mmu-miR-297a-3p, mmu-miR-129-5p and mmu-miR-107-3p in the 4 hour 10 μm Cd treatment group were basically the same as that of the chip result. Our results indicate that Cd exposure can affect alternative splicing of the kitl pre-mRNA in ovarian granulosa cells, and miRNAs play regulatory roles in the alternative splicing of kitl.

Toxic-Metal-Induced Alteration in miRNA Expression Profile as a Proposed Mechanism for Disease Development

“…Ovaries are one organ known to be susceptible to Cd accumulation and have a greater potential risk for toxicity either before or during pregnancy [55]. In ovarian granulosa cells, a 4-h exposure to Cd resulted in altered expression of five miRNAs involved in the kit ligand (kitl ) pre-mRNA alternative splicing [56]. Cd has been identified as a possible causative agent for preeclampsia, in which the ability of Cd to bioaccumulate increases its potential toxicity.…”

Peer Review #2 of "Using bioinformatics and metabolomics to identify altered granulosa cells in patients with diminished ovarian reserve (v0.1)"

“…MiR-107 targets 21 DEGs, including five steroidogenic genes: HMGCS1, FDFT1, CYP51A1, SQLE, and EBP. miRNA-107 expression in murine ovarian GCs exposed to cadmium was significantly different from expression in the control group, and miRNA-107 can regulate kit ligand (kitl) expression (Wang et al, 2018). Kitl plays an important role in the recruitment of primitive follicles (Parrott & Skinner 1999), the proliferation and differentiation of GCs, the recruitment of theca cells, and early steroid hormone synthesis (Flanagan et al, 1991).…”

Alternative Splicing in the Hippo Pathway—Implications for Disease and Potential Therapeutic Targets

“…It is also possible that external and environmental factors may be linked to AS that can affect the Hippo pathway. A recent paper from the Zhang group [ 84 ] shows that the toxic metal cadmium, which can enter the body through contaminated water, air and food, and can readily accumulate in the body and particularly the ovaries, can affect kit ligand ( KITL ) pre-mRNA AS of exon 6 changing the ratio of kitl1/kitl2 mRNA. Treatment of murine ovarian cells with cadmium was shown to affect the expression of 29 microRNAs (miRNAs) associated with the KITL gene [ 84 ].…”

“…A recent paper from the Zhang group [ 84 ] shows that the toxic metal cadmium, which can enter the body through contaminated water, air and food, and can readily accumulate in the body and particularly the ovaries, can affect kit ligand ( KITL ) pre-mRNA AS of exon 6 changing the ratio of kitl1/kitl2 mRNA. Treatment of murine ovarian cells with cadmium was shown to affect the expression of 29 microRNAs (miRNAs) associated with the KITL gene [ 84 ]. Gene ontology analysis showed that the target genes of these 29 miRNAs were enriched in biological processes such as cellular metabolism, signal transduction, cell cycle and proliferation, differentiation and migration.…”

“…Gene ontology analysis showed that the target genes of these 29 miRNAs were enriched in biological processes such as cellular metabolism, signal transduction, cell cycle and proliferation, differentiation and migration. Interestingly, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that some of the target genes of these 29 miRNAs were enriched in the Hippo pathway, providing new evidence that environmental factors can affect the Hippo pathway by modulating AS [ 84 ].…”