Liu Libangxi, Sun Hong, Zhang Yang, Liu Chang, Zhuang Yong, Liu Miao, Ai Xuezheng, Long Dan, Huang Bo, Li Changqing, Zhou Yue, Dong Shiwu, Feng Chencheng
Department of Orthopaedics, Xinqiao Hospital Army Medical University Chongqing China.
Department of Orthopaedics Affiliated Hospital of Guizhou Medical University Guiyang Guizhou China.
JOR Spine. 2024 Jan 25;7(1):e1316. doi: 10.1002/jsp2.1316. eCollection 2024 Mar.
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 mA modification in nucleus pulpous (NP) tissues of rats at different ages.
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.
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.
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.
椎间盘（IVD）老化过程中N6-甲基腺苷（m6A）动态变化在很大程度上仍不清楚。本研究旨在探讨不同年龄大鼠髓核（NP）组织中m6A修饰的分布和模式。
进行组织学染色和磁共振成像（MRI）以评估IVD退变情况。采用实时定量聚合酶链反应（qRT-PCR）和蛋白质免疫印迹法分析m6A修饰因子的表达。随后，进行甲基化RNA免疫沉淀下一代测序和RNA测序，以鉴定NP组织中m6A甲基化组和转录组的差异。
与2月龄大鼠相比，我们发现20月龄大鼠NP组织中整体m6A水平以及甲基转移酶样蛋白3（Mettl3）和脂肪量和肥胖相关蛋白（FTO）的表达有显著变化。在NP老化过程中，931个基因中有1126个持续存在差异m6A峰，以及51个持续存在差异表达基因。基因本体（GO）和京都基因与基因组百科全书（KEGG）分析表明，这些m6A峰和m6A修饰基因主要参与椎间盘退变（IDD）的生物学过程和通路，如细胞外基质代谢、血管生成、炎症反应、雷帕霉素靶蛋白（mTOR）和腺苷酸活化蛋白激酶（AMPK）信号通路。同时，联合分析和维恩图显示，与2月龄和10月龄大鼠相比，共有405个衰老相关基因在20月龄大鼠中具有显著的甲基化和表达水平。此外，发现四个具有高甲基化修饰的衰老相关基因，包括BUB1、碳酸酐酶12（CA12）、含血小板反应蛋白基序的解聚蛋白样金属蛋白酶1（Adamts1）和含血小板反应蛋白基序的解聚蛋白样金属蛋白酶4（Adamts4）在蛋白质水平呈现差异表达，其中BUB1和CA12在NP老化过程中表达降低，而Adamts1和Adamts4表达升高。
总体而言，本研究阐明了IVD老化过程中m6A修饰的分布和模式。此外，m6A修饰基因参与了与IDD相关的生物学过程和通路。这些发现可能从衰老角度为IDD的机制和治疗提供新的见解。
