摘要
目的探讨长链非编码RNA(lncRNA)H19调控微小RNA⁃155(miR⁃155)在氯化锰(MnCl2)诱导SH⁃SY5Y细胞损伤中的作用机制。方法用MnCl2处理SH⁃SY5Y细胞并将H19过表达质粒(pcDNA3.1⁃H19)及阴性对照质粒(pcDNA3.1)、H19小干扰RNA(si⁃H19)及乱序无意义阴性对照(si⁃NC)、miR⁃155寡核苷酸模拟物(miR⁃155)及阴性对照(miR⁃NC)分别转染进细胞。采用四唑盐比色法(MTT)检测细胞存活率,流式细胞术检测细胞凋亡率,蛋白免疫印迹(Western blot)检测B淋巴细胞瘤-2(Bcl⁃2)、B淋巴细胞瘤-2相关X蛋白(Bax)和裂解的天冬氨酸特异性半胱氨酸蛋白酶3(cleaved Caspase⁃3)蛋白表达水平,酶联免疫吸附试验(ELISA)检测炎症因子白细胞介素⁃1β(IL⁃1β)、白细胞介素⁃6(IL⁃6)和肿瘤坏死因子α(TNF⁃α)分泌量,实时荧光定量PCR(qRT⁃PCR)检测H19和miR⁃155表达水平,双荧光素酶报告基因实验验证H19与miR⁃155的靶向调控关系。结果用MnCl2(250、500和1000μmol/L)处理SH⁃SY5Y细胞24 h可明显降低细胞存活率、Bcl⁃2蛋白表达水平和H19表达水平;明显增加细胞凋亡率,Bax和cleaved Caspase⁃3蛋白表达水平,IL⁃1β、IL⁃6和TNF⁃α分泌量,miR⁃155表达水平,且呈剂量依赖性(P<0.05)。H19靶向抑制miR⁃155的表达,H19过表达可逆转MnCl2对SH⁃SY5Y细胞增殖、凋亡和炎症因子分泌的影响(P<0.05),抑制H19表达或miR⁃155过表达可加剧MnCl2对SH⁃SY5Y细胞增殖、凋亡和炎症因子分泌的影响(P<0.05)。结论MnCl2诱导SH⁃SY5Y细胞损伤可能与H19调控miR⁃155进而促进炎症反应有关。
Objective To investigate the mechanism of long non⁃coding RNA(lncRNA)H19 regulating microRNA⁃155(miR⁃155)in manganese chloride⁃induced SH⁃SY5Y cell injury.Methods SH⁃SY5Y cells were treated with manganese chloride and H19 overexpression plasmid(pcDNA3.1⁃H19),negative control plasmid(pcDNA3.1),H19 small interfering RNA(si⁃H19),disordered nonsense negative control(si⁃NC),miR⁃155 oligonucleotide mimics(miR⁃155)and negative control(miR⁃NC)were transfected into the cells,respectively.Tetrazolium salt colorimetry(MTT)was used to detect the survival rate of the cells.Flow cytometry was used to detect the apoptosis rate.Western blot was used to detect the protein expression levels of B⁃lymphocytoma⁃2(Bcl⁃2),B⁃lymphocytoma⁃2⁃associated X protein(Bax)and cleaved aspartate⁃specific cysteine proteinase 3(cleaved Caspase⁃3).Enzyme⁃linked immunosorbent assay(ELISA)was used to detect secretion of inflammatory cytokines interleukin⁃1β(IL⁃1β),interleukin⁃6(IL⁃6)and tumor necrosis factor-α(TNF⁃α).Real⁃time quantitative PCR(qRT⁃PCR)was used to detect the expression levels of H19 and miR⁃155.Dual luciferase reporter gene assay was used to verify the targeting relationship between H19 and miR⁃155.Results Treatment of SH⁃SY5Y cells with manganese chloride(250,500,1000μmol/L)for 24 h significantly decreased cell survival rate,Bcl⁃2 protein expression level and H19 expression level,and significantly increased the apoptosis rate,Bax and cleaved caspase⁃3 protein expression levels,IL⁃1β,IL⁃6,TNF⁃αsecretion levels and miR⁃155 expression levels,which were dose⁃dependent(P<0.05).H19 targeted inhibition of the expression of miR⁃155.H19 overexpression reversed the effects of manganese chloride on the proliferation,apoptosis and secretion of inflammatory factors(P<0.05),inhibiting the expression of H19 or overexpression of miR⁃155 aggravated the effects of manganese chloride on the proliferation,apoptosis and secretion of inflammatory factors(P<0.05).Conclusi
作者
刘萍萍
庄建发
纪荣佳
王俊毅
吴进
陈大朝
LIU Ping-ping;ZHUANG Jian-fa;JI Rong-jia;WANG Jun-yi;WU Jin;CHEN Da-chao(The Ninth Hospital of the Joint Service Support Force of the People’s Liberation Army of China/Southeast Hospital Affiliated to Xiamen University,Zhangzhou,Fujian 363000,China;Medical College of Xiamen University,Xiamen,Fujian 361102,China)
出处
《毒理学杂志》
CAS
2023年第4期289-296,共8页
Journal of Toxicology
基金
国家自然科学基金(81402217)。
