HSPA13在ARPE19细胞氧化应激中的作用  

The role of HSPA13 in oxidative stress of human retinal pigment epithelial ARPE19 cells

作　　者：李梦雯 吕亚莉 徐国彤 吕立夏[1,2] LI Meng-wen;LU Ya-li;XU Guo-tong;LU Li-xia(Dept.of Biochemistry and Molecular Biology Research,Regenerative Medicine,Tongji University School of Medicine,Shanghai 200092,China;Dept.of Ophthalmology,Tenth People s Hospital,Tongji University School of Medicine,Shanghai 200072,China)

机构地区：[1]同济大学医学院再生医学系生物化学与分子生物学教研室,上海200092 [2]同济大学附属第十人民医院眼科,上海200072

出　　处：《同济大学学报（医学版）》2019年第5期554-562,共9页Journal of Tongji University(Medical Science)

基　　金：国家重点基础研究发展计划(973计划)(2013CB967501);上海市自然科学基金(17ZR1431300)

摘　　要：目的探讨HSPA13在ARPE19细胞氧化应激中的作用。方法采用CCK8法检测不同浓度H2O2对ARPE19细胞活力的影响;Western印迹法和免疫荧光检测H2O2对HSPA13表达的影响;CCK8法检测HSPA13的表达水平对氧化应激诱导的细胞活力的影响;过表达或siRNA干扰HSPA13后,分别检测氧化应激相关指标和促炎因子的分泌;RNA测序分析空载组和HSPA13干扰组差异表达的基因,并进行GO富集及KEGG信号通路分析。结果CCK8法结果显示,与H2O2空白对照组相比,100、200、300、400μmol/L H2O2处理组细胞活力降低(P<0.05)。Western印迹法结果显示,与H2O2空白对照组相比,H2O2处理组HSPA13的表达上升(P<0.05)。CCK8法结果显示,过表达HSPA13可保护细胞活力。氧化应激相关指标和促炎因子检测结果显示,与H2O2处理组相比,过表达HSPA13加H2O2处理组中SOD1和GPX1 mRNA表达上升,MDA含量降低,GSH含量上升,IL-8、IL-2和IL-1β分泌下降;而siRNA干扰HSPA13加H2O2处理组的结果则相反,并且活性氧类含量上升(P<0.05)。RNA测序结果显示,与空载组相比,HSPA13干扰组差异基因表达上调的有64个,下调的有254个。GO富集分析表明,HSPA13与细胞代谢或多种生理过程相关。KEGG分析显示,HSPA13与PI3K-Akt信号通路、TGFβ信号通路相关。结论氧化应激诱导ARPE19细胞中HSPA13的表达上调;HSPA13在细胞氧化损伤中发挥保护作用,这为寻找年龄相关性黄斑变性新的治疗靶点提供有益的线索。Objective To investigate the role of HSPA13 in oxidative stress of human retinal pigment epithelial ARPE19 cells.Methods The effect of different concentrations of H2O2 on cell viability was detected by CCK8 assay.And the expression of HSPA13 was detected by Western blotting and immunofluorescence,respectively.ARPE19 cells were transfected with pEGFP-N2-HSPA13 plasmid or siHSPA13 for over-expression or inhibited expression of HSPA13,the oxidative stress-related indicators and secretion of pro-inflammatory factors were detected.The differentially expressed genes between control group and HSPA13 interference group were examined with RNA-sequence,and analyzed with GO enrichment and KEGG signaling pathway.Results CCK8 assay showed that compared with H2O2 blank control group,the cell viability of 100μmol/L,200μmol/L,300μmol/L,and 400μmol/L H2O2 treatment groups were decreased significantly(P<0.05).Western blotting showed that the expression level of HSPA13 in H2O2 treatment group was significantly higher than that in H2O2 blank control group(P<0.05).CCK8 results showed that overexpression of HSPA13 protected cell viability.Compared with the H2O2 treatment group,the expression of SOD1 and GPX1 mRNA were increased,the content MDA was decreased,the secretion of IL-8,IL-2 and IL-1βwere decreased in the over-expressing HSPA13 plus H2O2 treatment group;and the results were reversed in the siRNA interference HSPA13 plus H2O2 treatment group(P<0.05).The RNA-sequence results showed that compared with control group,64 genes were up-regulated and 254 genes were down-regulated.GO enrichment analysis indicated that HSPA13 was associated with cellular metabolism or multiple physiological processes.KEGG analysis revealed that HSPA13 was associated with the PI3K-Akt signaling pathway and the TGFβsignaling pathway.Conclusion The expression of HSPA13 is up-regulated in oxidative stress-induced human retinal pigment epithelial ARPE19 cells.HSPA13 plays a protective role in oxidative damage,which may provide a useful clue of new the

关 键 词：过氧化氢 氧化应激 应激伴侣蛋白 视网膜色素上皮细胞 年龄相关性黄斑变性 

分 类 号：R774.5[医药卫生—眼科]