Background The N6-methyladenosine (m6A) dynamics in the progression of intervertebral disc (IVD) aging remain largely unknown. This study aimed to explore the distribution and pattern of m 6 A modification in nucleus pulpous (NP) tissues of rats at different ages. Methods Histological staining and MRI were performed to evaluate the degeneration of IVD. The expression of m6A modifiers was analyzed using qRT-PCR and western blot. Subsequently, methylated RNA immunoprecipitation next generation sequencing and RNA-seq were conducted to identify differences in m6A methylome and transcriptome of NP tissues. Results Compared to 2-month-old rats, we found significant changes in the global m6A level and the expression of Mettl3 and FTO in NP tissues from 20-month-old rats. During the progression of NP aging, there were 1126 persistently differentially m6A peaks within 931 genes, and 51 persistently differentially expressed genes. GO and KEGG analyses showed that these m6A peaks and m6A modified genes were mainly engaged in the biological processes and pathways of intervertebral disc degermation (IDD), such as extracellular matrix metabolism, angiogenesis, inflammatory response, mTOR and AMPK signaling pathways. Meanwhile, conjoint analyses and Venn diagram revealed a total of 405 aging related genes contained significant methylation and expression levels in 20-month-old rats in contrast to 2-month-old and 10-month-old rats. Moreover, it was found that four aging related genes with hypermethylated modification including BUB1, CA12, Adamts1, and Adamts4 depicted differentially expressed at protein level, of which BUB1 and CA12 were decreased, while Adamts1 and Adamts4 were increased during the progression of NP aging. Conclusion Collectively, this study elucidated the distribution and pattern of m6A modification during the aging of IVD. Furthermore, the m6A modified genes were involved in the IDD related biological processes and pathways. These findings may provide novel insights into the mechanisms and therapies of IDD from the perspective of aging.
Wang Yue,Fang Xi,Yin Huanshun,Zhou Yunlei,Yang Yue,Ai Shiyun
A novel photoelectrochemical immunosensor has been constructed for the determination of methylated RNA. MoS 2 nanosheets with large specific area were employed as photoactive material, gold nanoparticles were used as signal amplification unit and immobilization matrix of 4-mercaptophenylboronic acid, anti-m 6 A antibody was adopted as methylated RNA recognition reagent, and poly(U) polymerase–mediated RNA chain extension and Ru(NH 3 ) 6 3+ were used as assisted signal amplification unit. With the sensitization effect of Ru(NH 3 ) 6 3+ , the photoactivity of WS 2 nanosheets was improved greatly, which also improved the sensitivity. Using visible-light excitation and ascorbic acid as electron donor, the sensitive determination of methylated RNA was achieved by monitoring the photocurrent change with different concentrations of methylated RNA. This photoelectrochemical immunosensor has a wide linear relationship with methylated RNA concentration from 0.05 to 35 nM under optimal experimental conditions. The low detection limit of 14.5 pM was realized based on 3σ criterion. In addition to the good selectivity, this sensor also presents high reproducibility with a relative standard deviation of 1.4% for the photocurrent of seven electrodes. The applicability of the developed method was also investigated by detecting the level of methylated RNA in corn seedling leaves with and without sulfadiazine treatment.
Graphical abstract A novel photoelectrochemical immunosensor was developed for methylated RNA detection using the photoactive material of MoS2 and poly(U) polymerase–mediated RNA chain extension.
危险品化学品经营许可证（带存储）