生物信息学分析萝卜硫素上调RAB7交互溶酶体蛋白与非小细胞肺癌细胞自噬的关联

Association between sulforaphane-upregulated RAB7 interacting lysosomal protein and autophagy of non-small cell lung cancer cell by bioinformatics analysis

目的研究萝卜硫素(sulforaphane,SFN)抑制非小细胞肺癌(non-small cell lung cancer,NSCLC)细胞自噬溶酶体形成的机制。方法利用免疫荧光共聚焦技术观察溶酶体在非小细胞肺癌细胞A549中的亚细胞定位;使用高效液相色谱与质谱联用分析SFN(20μmol/L)处理A549细胞后蛋白质组的表达差异;分别使用不同浓度的SFN(0,10,20,30μmol/L)处理人非小细胞肺癌A549细胞,通过蛋白免疫印迹技术检测RILP蛋白的表达。利用TCGA及UNLCAN数据库分析肺癌组织与癌旁组织中RAB7交互溶酶体蛋白(RILP)及ATP6V0D1蛋白的差异表达;利用ONCOLNC数据库分析RILP及ATP6V0D1与肺癌患者生存期的关联;利用TCGA数据库分析在肺腺癌和肺鳞癌组织中RILP与自噬相关蛋白LC3Ⅱ、RILP与ATP6V0D1、ATP6V0D1与LC3Ⅱ的相关性;利用STRING数据库分析RILP蛋白与微管蛋白TUBB/TUBA1A、LC3Ⅱ间的相互作用。结果在SFN处理的A549细胞中观察到溶酶体发生核周聚集。液相色谱和质谱联用分析表明:在SFN处理的A549细胞中,380个蛋白表达上调,278个蛋白表达下调,差异有统计学意义(P<0.05)。蛋白免疫印迹结果显示:随着SFN浓度(0,10,20,30μmol/L)的升高,RILP的表达显著上升(P<0.05)。TCGA数据库结果显示:RILP、ATP6V0D1蛋白在非小细胞肺癌组织中低表达(P<0.05)。与高表达RILP、ATP6V0D1蛋白的患者相比,低表达RILP、ATP6V0D1蛋白的肺癌患者生存期短(P<0.05)。STRING数据库结果显示RILP蛋白与微管蛋白TUBB/TUBA1A以及自噬相关蛋白LC3Ⅱ相互作用。同时,TCGA数据库显示在肺腺癌和肺鳞癌组织中,RILP蛋白与ATP6V0D1蛋白的表达呈正相关(P<0.01),RILP蛋白与LC3Ⅱ蛋白的表达呈正相关(P<0.01),ATP6V0D1蛋白与LC3Ⅱ蛋白的表达呈正相关(P<0.01)。结论SFN通过上调RILP的表达,从而加强RILP与溶酶体ATP6V0D1蛋白及LC3Ⅱ的关联,抑制非小细胞肺癌细胞自噬溶酶体形成。

Objective To study the mechanism of sulforaphane(SFN)inhibiting autolysosome formation in human non-small cell lung cancer(NSCLC)cells.Methods Immunofluorescence confocal was used to observe the subcellular localization of lysosomes in NSCLC A549 cells.The proteome expression was analyzed in A549 cells after treated with SFN(20μmol/L)by high performance liquid chromatography and mass spectrometry(HPLC-MS/MS).A549 cells were treated with different concentrations of SFN(0,10,20,30μmol/L),and the expression of RILP protein was detected by Western blot.The differential expression levels of RAB7 cross-lysosomal protein(RILP)and ATP6V0D1 protein in lung cancer and adjacent tissues were analyzed based on TCGA and UNLCAN databases.The association between RILP,ATP6V0D1 and survival of lung cancer patients was analyzed using ONCOLNC database.The correlation between RILP and LC3Ⅱ,between RILP and ATP6V0D1,between ATP6V0D1 and LC3Ⅱin lung adenocarcinoma and lung squamous cell carcinoma tissues were analyzed using TCGA database.The interaction of RILP,TUBB/TUBA1A and LC3Ⅱwas analyzed using STRING database.Results Perinuclear aggregation of lysosomes was observed in SFN-treated A549 cells.HPLC-MS/MS analysis showed that 380 proteins were upregulated and 278 proteins were downregulated in SFN-treated A549 cells(P<0.05).Western blot results showed that the expression of RILP was dose-dependently increased after treated with 0,10,20,30μmol/L SFN(P<0.05).TCGA database results showed that RILP and ATP6V0D1 were lowly expressed in NSCLC tissues(P<0.05).The survival time was shorter in lung cancer patients with low expression of RILP and ATP6V0D1 than that of patients with high expression of RILP and ATP6V0D1(P<0.05).STRING database results showed that RILP was interacted with TUBB/TUBA1A and autophagy-related protein LC3Ⅱ.TCGA database showed that RILP was positively correlated with ATP6V0D1 expression in lung adenocarcinoma and lung squamous cell carcinoma(P<0.01),the expression of RILP was positively correlated with LC3Ⅱ(P<0.01),and the expression of ATP6V0D1 was positively correlated with LC3Ⅱ(P<0.01).Conclusion SFN strengthens the association between lysosomal ATP6V0D1 and LC3Ⅱ,and inhibits the formation of autolysosome in NSCLC cells by up-regulating the expression of RILP.