目的：探究miR-1246调控甲基转移酶样3(METTL3)介导的沉默信息调节因子1(SIRT1)N⁶-甲基腺苷(m⁶A)修饰对高糖诱导的视网膜微血管内皮细胞(RMECs)损伤的影响。

方法：双荧光素酶实验检测miR-1246调控METTL3表达； RMECs细胞分为对照组、模型(HG)组、高糖+敲低对照(HG+anti-miR-NC)组、高糖+敲低miR-1246表达(HG+anti-miR-1246)组、高糖+过表达对照(HG+NC)组、高糖+过表达METTL3(HG+METTL3)组、高糖+过表达miR-1246+对照(HG+miR-1246+NC)组、高糖+过表达miR-1246+METTL3(HG+miR-1246+METTL3)组。经过高糖诱导48 h后，CCK-8法检测细胞存活； Annexin V-FITC/PI法检测细胞凋亡； Transwell实验检测细胞迁移和侵袭； ELISA法检测细胞氧化应激和炎症水平； 比色法检测总RNA中m⁶A甲基化水平； MeRIP-qPCR法检测SIRT1 m⁶A甲基化水平； 实时荧光定量PCR检测细胞miR-1246、METTL3、SIRT1 mRNA表达； Western blot检测细胞METTL3、SIRT1及内皮-间充质转化(EndMT)标志物蛋白表达。

结果：miR-1246调控METTL3表达。与对照组比较，HG组细胞存活率降低，凋亡率升高，迁移和侵袭细胞数增加，细胞培养上清液乳酸脱氢酶(LDH)活性、肿瘤坏死因子-α(TNF-α)、白细胞介素(IL)-6水平升高，IL-10水平降低，细胞丙二醛(MDA)水平升高，超氧化物歧化酶(SOD)活性降低，细胞miR-1246表达升高，总RNA m⁶A水平和SIRT1 m⁶A水平降低，METTL3、SIRT1、分化群抗原31(CD31)、血管内皮钙黏蛋白(VE-cadherin)表达降低，波形蛋白(Vimentin)、Snail同源物1(Snail1)表达升高(均P<0.05)； 与HG+anti-miR-NC组比较，HG+anti-miR-1246组细胞存活率升高，凋亡率降低，迁移和侵袭细胞数减少，细胞培养上清液LDH活性、TNF-α、IL-6水平降低，IL-10水平升高，细胞MDA水平降低，SOD活性升高，细胞miR-1246表达降低，总RNA m⁶A水平和SIRT1 mRNA m⁶A水平升高，METTL3、SIRT1、CD31、VE-cadherin表达升高，Vimentin、Snail1表达降低(均P<0.05)； 与HG+NC组比较，HG+METTL3组细胞存活率升高，凋亡率降低，迁移和侵袭细胞数减少，细胞培养上清液LDH活性、TNF-α、IL-6水平降低，IL-10水平升高，细胞MDA水平降低，SOD活性升高，细胞miR-1246表达降低，总RNA m⁶A水平和SIRT1 mRNA m⁶A水平升高，METTL3、SIRT1、CD31、VE-cadherin表达升高，Vimentin、Snail1表达降低(均P<0.05)； 与HG+miR-1246+NC组比较，HG+miR-1246+METTL3组细胞存活率升高，凋亡率降低，迁移和侵袭细胞数减少，细胞培养上清液LDH活性、TNF-α、IL-6水平降低，IL-10水平升高，细胞MDA水平降低，SOD活性升高，细胞miR-1246表达降低，总RNA m⁶A水平和SIRT1 mRNA m⁶A水平升高，METTL3、SIRT1、CD31、VE-cadherin表达升高，Vimentin、Snail1表达降低(均P<0.05)。

结论：miR-1246通过调控METTL3介导的SIRT1 m⁶A修饰，促进高糖诱导的RMECs细胞凋亡、侵袭转移、氧化应激、炎症反应及EndMT过程。

METHODS:Dual luciferase assay was used to detect miR-1246 regulation of METTL3 expression; RMECs cells were divided into control group, high glucose(HG)group, high glucose+knocking down control(HG+anti-miR-NC)group, high glucose+knocking down miR-1246 expression(HG+anti-miR-1246)group, high glucose+overexpression control(HG+NC)group, high glucose+overexpression METTL3(HG+METTL3)group, high glucose+overexpression miR-1246+control(HG+miR-1246+NC)group, and high glucose+overexpression miR-1246+METTL3(HG+miR-1246+METTL3)group. After induction of high glucose for 48 h, CCK-8 method was used to detect cell survival; Annexin V-FITC/PI method was used to detect cell apoptosis; Transwell experiment was used to detect cell migration and invasion; ELISA method was used to detect cell oxidative stress and inflammation levels; Colorimetric method was used to detect m⁶A methylation level in total RNA; MeRIP-qPCR method was used to detect SIRT1 m⁶A methylation level; Real-time quantitative PCR was used to detect miR-1246, METTL3, SIRT1 mRNA expression in cells; Western blot was used to detect METTL3, SIRT1 and endothelial mesenchymal transition(EndMT)markers protein expression in cells.

RESULTS: The MiR-1246 regulated METTL3 expression. Compared with the control group, cell survival rate was decreased in the HG group, apoptosis rate was increased, and the number of migrating and invading cells were increased, lactate dehydrogenase(LDH)activity, tumor necrosis factor-α(TNF-α), and interleukin(IL)-6 levels in cell culture supernatant were increased, IL-10 level was decreased, malondialdehyde(MDA)level was increased, superoxide dismutase(SOD)activity was decreased, miR-1246 expression was increased, total RNA m⁶A level and SIRT1 m⁶A level were decreased, METTL3, SIRT1, cluster of differentiation 31(CD31)and vascular endothelial cadherin(VE-cadherin)expression were decreased, while Vimentin and Snail1 expression were increased(all P<0.05); compared with the HG+anti-miR-NC group, cell survival rate was increased in the HG+anti-miR-1246 group, apoptosis rate was decreased, and the number of migrating and invading cells were decreased, LDH activity, TNF-α, and IL-6 levels in cell culture supernatant were decreased, IL-10 level was increased, MDA level was decreased, SOD activity was increased, miR-1246 expression was decreased, total RNA m⁶A level and SIRT1 m⁶A level were increased, METTL3, SIRT1, CD31 and VE-cadherin expression were increased, while Vimentin and Snail1 expression were decreased(all P<0.05); compared with the HG+NC group, cell survival rate was increased in the HG+METTL3 group, apoptosis rate was decreased, and the number of migrating and invading cells were decreased, LDH activity, TNF-α, and IL-6 levels in cell culture supernatant were decreased, IL-10 level was increased, MDA level was decreased, SOD activity was increased, miR-1246 expression was decreased, total RNA m⁶A level and SIRT1 m⁶A level were increased, METTL3, SIRT1, CD31 and VE-cadherin expression were increased, while Vimentin and Snail1 expression were decreased(all P<0.05); compared with the HG+miR-1246+NC group, cell survival rate was increased in the HG+miR-1246+METTL3 group, apoptosis rate was decreased, and the number of migrating and invading cells were decreased, LDH activity, TNF-α, and IL-6 levels in cell culture supernatant were decreased, IL-10 level was increased, MDA level was decreased, SOD activity was increased, miR-1246 expression was decreased, total RNA m⁶A level and SIRT1 m⁶A level were increased, METTL3, SIRT1, CD31 and VE-cadherin expression were increased, while Vimentin and Snail1 expression were decreased(all P<0.05).

CONCLUSION:The miR-1246 promotes high glucose-induced apoptosis, invasion and metastasis, oxidative stress, inflammatory response, and EndMT process in RMECs cells by regulating METTL3 mediated SIRT1 m⁶A modification.