N6-methyladenosine modification of PLOD2 causes spermatocyte damage in rats with varicocele

In recent years, N6-methyladenosine (m6A) methylation modification of mRNA has been studied extensively. It has been reported that m6A determines mRNA fate and participates in many cellular functions and reactions, including oxidative stress. The PLOD2 gene encodes a protein that plays a key role in tissue remodeling and fibrotic processes. The m6A methylation and expression levels of PLOD2 were determined by m6A methylated RNA immunoprecipitation sequencing (MeRIP-seq) and MeRIP-quantitative polymerase chain reaction (qPCR) in the testes of varicocele rats compared with control. To determine whether IGF2BP2 had a targeted effect on the PLOD2 mRNA, RNA immunoprecipitation-qPCR (RIP-qPCR) and luciferase assays were performed. CRISPR/dCas13b-ALKBH5 could downregulate m6A methylation level of PLOD2, which plays an important role in PLOD2-mediated cell proliferation and apoptosis in GC-2 cells. PLOD2 was frequently exhibited with high m6A methylation and expression level in the testes of varicocele rats compared with control. In addition, we found that IGF2BP2 binds to the m6A-modified 3′ untranslated region (3′-UTR) of PLOD2 mRNA, thereby positively regulating its mRNA stability. Targeted specific demethylation of PLOD2 m6A by CRISPR/dCas13b-ALKBH5 system can significantly decrease the m6A and expression level of PLOD2. Furthermore, demethylation of PLOD2 mRNA dramatically promote GC-2 cell proliferation and inhibit cell apoptosis under oxidative stress. As a result, we found that varicocele-induced oxidative stress promoted PLOD2 expression level via m6A methylation modification. In addition, targeting m6A demethylation of PLOD2 by CRISPR/dCas13b-ALKBH5 system can regulate GC-2 cell proliferation and apoptosis under oxidative stress. Taken together, our study has acquired a better understanding of the mechanisms underlying male infertility associated with oxidative stress, as well as a novel therapeutic target for male infertility.