Qin Yongzhang, Wu Suzhen, Zhang Fengxia, Zhou Xueyan, You Cong, Tan Fei
Department of Endocrinology, First Affiliated Hospital of Gannan Medical University, Ganzhou, Jiangxi, China.
Ganzhou Key Laboratory of Thyroid Cancer, First Affiliated Hospital of Gannan Medical University, Ganzhou, Jiangxi, China.
Environ Toxicol. 2023 Nov;38(11):2772-2782. doi: 10.1002/tox.23917. Epub 2023 Aug 8.
Diabetic nephropathy (DN) is a major cause of end-stage renal disease throughout the world, and m6A modification plays a critical role in the progression of DN. We aimed to find m6A-related genes and their regulatory mechanisms in DN.
The expression levels of four important m6A-related genes (METTL16, RBM15, IGF2BP1, and ALKBH5) were detected by quantitative real-time PCR (RT-qPCR). RBM15 was chosen and its function was explored. The downstream pathway of RBM15 was screened by transcriptome sequencing. The levels of AGE, inflammation, and oxidative stress were determined with enzyme-linked immunosorbent assay, and the expression of AGE-RAGE pathway-related proteins were detected by Western blot (WB). Cell proliferation was assessed by Cell counting Kit-8 (CCK-8). The levels of pyroptosis-related proteins were evaluated by RT-qPCR or WB.
METTL16 and RBM15 were up regulated in the mouse model of DN, in which RBM15 was more significant. Silencing RBM15 recovered cell proliferation, reduced the levels of inflammation factors, and inhibited cell pyroptosis in high glucose-induced HK-2 cells. Transcriptome sequencing suggested that the AGE-RAGE pathway might be downstream of RBM15. RBM15 knockdown reduced AGE level and the expression of AGE-RAGE pathway-related proteins. After silencing RBM15, we found that activating the AGE-RAGE pathway inhibited cell proliferation, increased the levels of inflammation factors, promoted oxidative stress, and induced cell pyroptosis in HK-2 cell model of DN.
The m6A-related gene RBM15 inhibited cell proliferation, promoted inflammation, oxidative stress, and cell pyroptosis, thereby facilitating the progression of DN through the activation of the AGE-RAGE pathway.
糖尿病肾病（DN）是全球终末期肾病的主要病因，且m6A修饰在DN进展中起关键作用。我们旨在寻找DN中与m6A相关的基因及其调控机制。
通过定量实时PCR（RT-qPCR）检测四个重要的m6A相关基因（METTL16、RBM15、IGF2BP1和ALKBH5）的表达水平。选择RBM15并探索其功能。通过转录组测序筛选RBM15的下游通路。用酶联免疫吸附测定法测定晚期糖基化终末产物（AGE）、炎症和氧化应激水平，并用蛋白质免疫印迹法（WB）检测AGE-RAGE通路相关蛋白的表达。通过细胞计数试剂盒-8（CCK-8）评估细胞增殖。通过RT-qPCR或WB评估细胞焦亡相关蛋白的水平。
在DN小鼠模型中，METTL16和RBM15上调，其中RBM15上调更显著。沉默RBM15可恢复高糖诱导的HK-2细胞的增殖能力，降低炎症因子水平，并抑制细胞焦亡。转录组测序表明AGE-RAGE通路可能是RBM15的下游通路。敲低RBM15可降低AGE水平及AGE-RAGE通路相关蛋白的表达。沉默RBM15后，我们发现在DN的HK-2细胞模型中，激活AGE-RAGE通路会抑制细胞增殖，增加炎症因子水平，促进氧化应激，并诱导细胞焦亡。
m6A相关基因RBM15抑制细胞增殖，促进炎症、氧化应激及细胞焦亡，从而通过激活AGE-RAGE通路促进DN的进展。
