ALKBH5对紫外线诱导的晶状体上皮细胞氧化损伤模型中DNA损伤修复的影响

目的　探究N⁶-甲基腺苷（m⁶A）去甲基化酶ALKBH5对紫外线诱导的晶状体上皮细胞（LEC）氧化损伤模型中DNA损伤修复的影响。方法　运用qRT-PCR和免疫印迹实验检测年龄相关性白内障（ARC）患者和对照组晶状体前囊膜上皮细胞中ALKBH5的mRNA和蛋白的表达。通过紫外线B（UVB）构建晶状体上皮细胞株SRA01/04细胞氧化损伤模型和靶向ALKBH5设计小干扰RNA（siRNA）转染构建敲降模型，运用qRT-PCR和免疫印迹实验检测氧化损伤模型和敲降模型中ALKBH5的mRNA和蛋白表达。运用CCK-8法检测Control组、UVB组、UVB+siNC组和UVB+siALKBH5#3组中SRA01/04细胞活力变化。免疫荧光染色检测UVB+siNC组和UVB+siALKBH5#3组中15A3的荧光强度变化。运用qRT-PCR检测转染对照siNC组和转染siALKBH5#3组中11个DNA氧化损伤修复基因（ODRGs）的mRNA表达变化。结果　在ARC患者的晶状体前囊膜上皮细胞中，ALKBH5的mRNA和蛋白表达水平均显著升高。在UVB以时间梯度诱导SRA01/04的细胞氧化损伤模型中，ALKBH5的mRNA和蛋白表达水平均呈上升后下降趋势，其中UVB照射10 min后ALKBH5 mRNA和蛋白表达升高最为显著。ALKBH5敲降效率结果显示，与转染对照siNC组相比，转染靶向ALKBH5的siRNA后，ALKBH5的mRNA和蛋白表达均显著下降。CCK-8法检测结果显示，与UVB+siNC组相比，UVB+siALKBH5#3组SRA01/04细胞活力明显降低。免疫荧光染色检测结果显示，与UVB+siNC组相比，UVB+siALKBH5#3组SRA01/04细胞内DNA氧化损伤指标15A3染色显著增加。同时，与转染对照siNC组相比，转染ALKBH5#3组SRA01/04细胞的ODRGs中，TREX1、FANCD2、LIG1、MSH2、MSH3、RPA2、SMUG1、XRCC6 mRNA的表达均显著上升，DCLRE1A mRNA的表达显著下降，MGMT和MRE11A mRNA的表达则未见明显差异。结论　m⁶A去甲基化酶ALKBH5在UVB诱导的LEC氧化损伤模型中诱导性表达上升，敲降ALKBH5可促进LEC内大部分ODRGs表达升高，参与调控LEC内损伤DNA的修复，阻止ARC的发生。

Effect of ALKBH5 on DNA repair in lens epithelial cells exposed to ultraviolet-induced oxidative damage

Objective　To investigate the effect of N6-methyladenosine (m⁶A)-demethylase ALKBH5 on the repair of damaged DNA in lens epithelial cells (LECs) exposed to ultraviolet (UV)-induced oxidative damage. Methods　The mRNA and protein levels of ALKBH5 in anterior LECs of age-related cataract (ARC) patients and controls were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot. The SRA01/04 oxidative damage model was constructed by UVB and the knockdown model was constructed by transfecting SRA01/04 cells with small interfering RNA (siRNA) targeting ALKBH5. The mRNA and protein levels of ALKBH5 in the oxidative damage and knockdown models were evaluated by qRT-PCR and Western blot. Cell viability in the control group, UVB group, UVB+siNC group, and UVB+siALKBH5#3 group was determined by CCK8. The fluorescence intensity of damaged DNA marker 15A3 in the UVB+siNC group and UVB+siALKBH5#3 group was measured by immunofluorescence staining. The mRNA expression of 11 DNA oxidative damage repair genes (ODRGs) in the transfection control siNC and transfection siALKBH5#3 groups was assessed by qRT-PCR. Results　The mRNA and protein levels of ALKBH5 dramatically increased in anterior LECs of ARC patients. The mRNA and protein levels of ALKBH5 in the UVB-induced oxidative damage model increased at first and then decreased. After 10 minutes of UVB radiation, the mRNA and protein levels of ALKBH5 increased most significantly. The knockdown efficiency of ALKBH5 revealed that the mRNA and protein levels of ALKBH5 notably decreased after transfection with siRNA targeting ALKBH5 when compared with the transaction control siNC group. CCK8 assay results showed that compared with the UVB+siNC group, cell viability in the UVB+siALKBH5#3 group was significantly reduced. Immunofluorescence staining results showed that compared with the UVB+siNC group, the fluorescence intensity of the DNA oxidative damage marker 15A3 in the UVB+siALKBH5#3 group significantly increased. Compared with the transfection control siNC group, the mRNA expression of TREX1, FANCD2, LIG1, MSH2, MSH3, RPA2, SMUG1, and XRCC6 among ODRGs in the transaction ALKBH5#3 group increased significantly, the mRNA expression of DCLRE1A decreased significantly, and the mRNA expression of MGMT and MRE11A showed no significant differences. Conclusion　The expression of m⁶A demethylase ALKBH5 increases in the UVB-induced LEC oxidative damage model. The knockdown of ALKBH5 can promote the expression of most ODRGs, regulate the repair of damaged DNA in LECs, and prevent the occurrence of ARC.