Aberrant mRNA 5-methylcytosine modification contributes to ovarian dysfunction in a rat model of polycystic ovary syndrome
Xiaohong Chen, Jing Lin, Zhoulin Chi, Sha Yang, Shuguang YuObjective
This study investigated the altered landscape of messenger RNA 5-methylcytosine (m5C) methylation and the functional involvement of m5C-related regulators in a rat model of polycystic ovary syndrome (PCOS).
Methods
A PCOS rat model was established by dehydroepiandrosterone (DHEA) induction, with the phenotype confirmed through estrous cycle monitoring, serum sex hormone analysis, and ovarian morphological assessment. Differentially m5C-methylated mRNAs were identified using an m5C epitranscriptomic microarray, and their associated biological pathways were predicted by KEGG analysis. Global m5C methylation levels and the expression and m5C enrichment of selected pathway-related genes were validated in ovarian tissues and isolated primary ovarian granulosa cells using dot blot, RT-qPCR, Western blotting, and methylated RNA immunoprecipitation quantitative PCR (MeRIP-qPCR). The functional roles of TET2 and NSUN6 were further evaluated using shRNA-mediated knockdown, followed by analysis of PI3K/AKT-related signaling proteins, serum sex hormones, and ovarian histopathology.
Results
m5C modifications were predominantly localized within coding sequences and 3′ untranslated regions. Among 1,090 RNAs showing both differential m5C methylation and expression, 36 genes exhibited significant correlations between methylation and expression changes. Global mRNA m5C methylation was significantly elevated in the PCOS group. The m5C-related regulators Nsun6 and Tet2 showed increased m5C enrichment and significantly elevated protein expression. KEGG analysis linked the differentially modified transcripts to endocrine resistance and insulin-related signaling pathways. Tet2, Nsun6, Src, Pi3k, and Akt transcripts exhibited increased m5C enrichment in PCOS ovarian tissues and primary granulosa cells. Concurrently, SRC and PI3K protein expression was increased, whereas AKT protein expression was decreased in the PCOS group. Knockdown of TET2 and NSUN6 was confirmed at the mRNA and protein levels and differentially modulated PI3K/AKT-related protein expression and phosphorylation status, as assessed by PI3K, p-PI3K, AKT, and p-AKT levels. TET2 and NSUN6 knockdown also partially improved PCOS-associated abnormalities in serum FSH, LH, the LH/FSH ratio, testosterone, estradiol, and ovarian morphology.
Conclusion
This study reveals a significantly altered mRNA m5C methylation landscape in a DHEA-induced PCOS rat model and primary ovarian granulosa cells. The functional knockdown data suggest that TET2 and NSUN6 may contribute to PCOS-associated ovarian dysfunction through differential regulation or remodeling of PI3K/AKT signaling. However, the opposite changes in the p-PI3K/PI3K and p-AKT/AKT ratios indicate that the pathway is differentially regulated rather than uniformly activated or inhibited. Because the DHEA model primarily mimics hyperandrogenism and MeRIP-qPCR does not provide single-nucleotide resolution, further validation in human samples and high-resolution m5C mapping is required to establish the precise molecular mechanism and clinical relevance of these findings.