摘要
目的:探讨N6-甲基腺嘌呤(N6-methyladenosine,m^(6)A)甲基化转移酶3(METTL3)在糖尿病性白内障发病中的作用机制。方法:用低糖和高糖培养基培养人晶状体上皮细胞系(SRA01/04)24h后,采用RT-qPCR和Western blot实验检测细胞的上皮-间质转分化(EMT)指标:E-钙黏蛋白(E-Cadherin)、N-钙黏蛋白(N-Cadherin)、紧密连接蛋白1(ZO-1)和α-平滑肌肌动蛋白(α-SMA)的变化情况;transwell和划痕实验检测细胞迁移能力。采用免疫荧光染色检测人晶状体前囊膜组织中METTL3的表达量及定位,m^(6)A dot blot实验检测在低糖和高糖培养基中培养24h细胞的m^(6)A甲基化水平,RT-qPCR和Western blot实验检测细胞中METTL3的RNA和蛋白表达量。加入METTL3抑制剂的培养基中培养24h的细胞,RT-qPCR和Western blot实验检测EMT指标的变化情况;m^(6)A dot blot实验检测细胞m^(6)A甲基化水平;Transwell和划痕实验检测细胞迁移能力。免疫荧光染色检测细胞中转化生成因子β(TGFβ1)的表达;RT-qPCR和Western blot实验检测细胞中的TGFβ1和SNAIL的表达量。结果:与低糖条件相比,高糖条件能够促进细胞EMT的发生,促进METTL3的表达和上调了细胞总RNA的m^(6)A甲基化水平(P<0.05)。高糖能够促进细胞的迁移能力。糖尿病性白内障患者晶状体前囊膜中METTL3表达较单纯年龄相关性白内障患者增高。与高糖+DMSO组相比,加入METTL3抑制剂STM2457,能够抑制细胞的EMT发生,抑制TGFβ1和SNAIL的表达,抑制细胞总RNA的m^(6)A甲基化水平(均P<0.05)。加入METTL3抑制剂STM2457后细胞迁移能力较高糖+DMSO组降低。结论:m^(6)A甲基化转移酶METTL3通过激活TGFβ1/SNAIL通路促进了在高糖条件下人晶状体上皮细胞的EMT发生从而诱导糖尿病性白内障的发生。
AIM:To investigate the role and mechanism of N6-methyladenosine(m^(6)A)methyltransferase 3(METTL3)in the pathogenesis of diabetic cataract.METHODS:We cultured SRA01/04 cells in low and high sugar media for 24h and measured changes in epithelial-mesenchymal transition(EMT)indicators(E-Cadherin,N-Cadherin,ZO-1 andα-SMA)using RT-qPCR and Western blot assays.Cell migration was also assessed using transwell and scratch assays.To investigate the expression level and localization of METTL3 in human lens anterior capsules tissues.Additionally,we used m^(6)A dot blot assay to detect the m^(6)A methylation level of cells cultured in low and high glucose media for 24h,and employed RT-qPCR and Western blot experiments to detect RNA and protein expression of METTL3 in cells.We then treated the cells with METTL3 inhibitor and measured changes in EMT markers by RT-qPCR and Western blot;m^(6)A methylation level was detected by m^(6)A dot blot test;cell migration was detected by Transwell.Finally,the expression of transforming growth factor-β(TGFβ1)in cultured cells was assessed by immunofluorescence staining and the expression levels of TGFβ1 and SNAIL in cells were determined using RT-qPCR and Western blot.RESULTS:Under high glucose conditions,the expression of EMT markers,METTL3,and m^(6)A methylation levels were significantly increased in cells(P<0.05).Furthermore,the migratory ability of cells was higher in high-sugar medium than in low-sugar medium.In human lens anterior capsules,METTL3 expression was higher in patients with diabetic cataract compared to those with age-related cataract.Importantly,treatment with the METTL3 inhibitor STM2457 inhibited EMT in cells,the expression of TGFβ1 and SNAIL,as well as m^(6)A methylation levels in cells(all P<0.05)compared to high-sugar+dimethyl sulfoxide(DMSO)group.Moreover,the migratory capacity of cells was reduced after the addition of STM2457 compared to the high-sugar+DMSO group.CONCLUSION:METTL3 promotes the EMT in human lens epithelial cells under high glucose conditions by activating the TGFβ1/SNAIL pathway,thus contributing to the development of diabetic cataracts.
作者
陈思
叶巍
唐韵
王文喆
葛轶睿
王雪莹
黄振平
Si Chen;Wei Ye;Yun Tang;Wen-Zhe Wang;Yi-Rui Ge;Xue-Ying Wang;Zhen-Ping Huang(School of Medicine,Southeast University,Nanjing 210003,Jiangsu Province,China;Department of Ophthalmology,General Hospital of Eastern Theater Command,Nanjing 210002,Jiangsu Province,China;Medical School of Nanjing University,Nanjing 210008,Jiangsu Province,China)
出处
《国际眼科杂志》
CAS
北大核心
2023年第8期1250-1259,共10页
International Eye Science
作者简介
陈思,女,在读硕士研究生,研究方向:白内障;通讯作者:黄振平,男,毕业于北京大学医学院,博士,主任医师,博士研究生导师,研究方向:白内障、角膜病、眼视光.hzp19633@hotmail.com。
