miR-181a-5p靶向DDIT4对UVB诱导的人皮肤成纤维细胞氧化损伤的影响OA
The Effect of miR-181a-5p Targeting DDIT4 on UVB-Induced Oxidative Damage in Human Skin Fibroblasts
目的:探讨微小 RNA-181a-5p(miR-181a-5p)是否可调控 DNA 损伤诱导转录物 4(DDIT4)影响中波紫外线(UVB)诱导的人皮肤成纤维细胞(HSF)氧化损伤.方法:以 30mJ/cm2 的UVB 照射 HSF,分别在第 1、3、5 和 7 天检测 miR-181a-5p 表达水平;将 HSF 分为对照(NC)组、UVB组、miR-181a-5p 低表达阴性对照(anti-NC)组、miR-181a-5p 低表达(anti-miR-181a-5p)组、anti-miR-181a-5p+DDIT4 低表达阴性对照(sh-NC)组和 anti-miR-181a-5p+DDIT4 低表达(sh-DDIT4)组;定量聚合酶链式反应(RT-PCR)检测细胞 miR-424-5p 和 DDIT4 mRNA 表达水平;细胞计数(CCK-8)检测细胞活力;流式细胞术检测细胞凋亡率;β-半乳糖苷酶染色法观察细胞衰老情况;检测细胞过氧化物歧化酶(SOD)、丙二醛(MDA)和过氧化氢酶(CAT)水平;蛋白免疫印迹法检测基质金属蛋白酶 1(MMP-1)、基质金属蛋白酶 9(MMP-9)、活化的半胱氨酸天冬氨酸蛋白酶-3(cleaved caspase-3)和DDIT4 蛋白表达水平;验证 miR-181a-5p 和 DDIT4 的靶向关系.结果:与对照组比较,UVB 照射组HSF 中 miR-181a-5p 表达水平明显升高(P<0.05);与 NC 组比较,UVB 组 HSF 中 miR-181a-5p 表达水平、凋亡率、衰老细胞比例、MDA 含量、MMP-1、MMP-9 和 cleaved caspase-3 蛋白表达水平升高,DDIT4 mRNA 表达水平、细胞活力、CAT 和 SOD 活性、DDIT4 蛋白表达水平降低(P<0.05);与 UVB 组和anti-NC 组比较,anti-miR-181a-5p 组 HSF 中 miR-181a-5p 表达水平、凋亡率、衰老细胞比例、MDA 含量、MMP-1、MMP-9 和 cleaved caspase-3 蛋白表达水平降低,DDIT4 mRNA 表达水平、细胞活力、CAT和 SOD 活性、DDIT4 蛋白表达水平升高(P<0.05);下调 DDIT4 表达可降低沉默 miR-181a-5p 表达对UVB 诱导的 HSF 氧化损伤的改善作用(P<0.05).结论:沉默 miR-181a-5p 表达可能通过提高 DDIT4表达来减轻 UVB 诱导的 HSF 氧化损伤.
Objective:To investigate whether microRNA-181a-5p(miR-181a-5p)can regulate DNA Damage Inducible Transcript 4(DDIT4)to affect Ultraviolet B(UVB)induced oxidative damage in human skin fibroblasts(HSF).Methods:HSF were irradiated with UVB at 30mJ/cm2,and the expression level of miR-181a-5p was detected on 1d,3d,5d,and 7d,respectively.HSF was separated into normal control(NC)group,the UVB group,the miR-181a-5p low-expression negative control(anti-NC)group,the miR-181a-5p low-expression(anti-miR-181a-5p)group,the anti-miR-181a-5p+DDIT4 low-expression negative control(sh-NC)group,and the anti-miR-181a-5p+DDIT4 low-expression(sh-DDIT4)group.Real-time polymerase chain reaction(RT-PCR)was used to detect the miR-424-5p and DDIT4 mRNA in cells.Cell Counting Kit-8(CCK-8)was performed to detect cell viability.Flow cytometry was performed to detect cell apoptosis rate.The β-galactosidase staining method was used to observe cellular aging.The malon-dialdehyde(MDA),superoxide dismutase(SOD),and Catalase(CAT)in cells were detected.Protein im-munoblotting was used to detect the Matrix Metalloproteinase 9(MMP-9),Matrix Metalloproteinase 1(MMP-1),Cleaved cysteine-aspartic acid protease-3(cleaved caspase-3),and DDIT4 proteins.The targeting relationship between miR-181a-5p and DDIT4 was validated.Results:Compared with the control group,the UVB group had higher miR-181a-5p in HSF(P<0.05).Compared with the NC group,the UVB group had higher miR-181a-5p,apoptosis rate,proportion of senescent cells,MDA content,MMP-1,MMP-9,and cleaved caspase-3 proteins in HSF,and lower DDIT4 mRNA,cell viability,CAT and SOD activities,and DDIT4 protein(P<0.05).Compared with the UVB group and anti-NC group,the anti-miR-181a-5p group had lower miR-181a-5p,apoptosis rate,proportion of senescent cells,MDA content,MMP-1,MMP-9,and cleaved caspase-3 proteins in HSF,and higher DDIT4 mRNA,cell viability,CAT and SOD activities,and DDIT4 protein(P<0.05).Downregulation of DDIT4 expression could reduce the improvement effect of silencing miR-181a-5p expression on UVB-induced HSF oxidative damage(P<0.05).Conclusion:Silen-cing miR-181a-5p expression may alleviate UVB-induced oxidative damage in HSF by enhancing DDIT4 ex-pression.
张名;黄燕;吴希晞
湖北省武汉市第三医院 整形外科,湖北 武汉 430061湖北省武汉市第三医院 整形外科,湖北 武汉 430061湖北省武汉市第三医院 整形外科,湖北 武汉 430061
DNA损伤诱导转录物4miR-181a-5p中波紫外线人皮肤成纤维细胞氧化损伤
DDIT4miR-181a-5pUVBHuman skin fibroblastsOxidative damage
《河北医学》 2026 (4)
614-621,8
武汉市卫健委医学科学研究项目,(编号:WX23A07)
评论