下调DDX39A对食管鳞癌细胞生物学行为的影响及其机制

Effect of DDX39A downregulation on biological behaviors of esophageal squamous cell carcinoma cells and its mechanism

目的探究DDX解旋酶39A(DEAD-box RNA helicases 39A,DDX39A)对食管鳞癌KYSE-150和TE-1细胞生物学行为的影响及作用机制。方法利用癌症基因组图谱(the cancer genome atlas,TCGA)和高通量基因表达数据库(gene expression omnibus,GEO)分析DDX39A在食管鳞癌肿瘤组织和正常组织中的表达差异。进一步在TCGA数据库中,采用Spearman相关分析(设置r>0.5,P<0.05)得到与DDX39A表达最相关基因,通过京都基因与基因组百科全书(Kyoto encyclopedia of genes and genomes,KEGG)和基因集富集分析(gene set enrichment analysis,GSEA),注释DDX39A在食管鳞癌中的生物学功能。通过慢病毒介导的短发夹RNA(shRNA)干扰技术下调DDX39A在食管鳞癌KYSE-150和TE-1细胞中的表达,并将细胞分为对照(shCtrl)组和DDX39A敲减(shDDX39A)组。利用qPCR和Western blot法检测敲减效率;利用Celigo法、克隆形成实验和MTT法检测KYSE-150和TE-1细胞的增殖能力和活力;利用流式细胞术检测KYSE-150和TE-1细胞的凋亡水平;利用Transwell实验检测KYSE-150和TE-1细胞的迁移和侵袭能力;利用裸鼠成瘤实验检测敲减DDX39A对体内肿瘤的作用。利用Western blot法筛选敲减DDX39A后KYSE-150细胞内蛋白表达水平显著变化的肿瘤相关经典通路分子。通过慢病毒转染法构建已筛选的分子CDH2(Cadherin 2)过表达的稳转细胞系,并将KYSE-150细胞分为shDDX39A+NC-OE组(转染shDDX39A慢病毒和过表达空载对照病毒)和shDDX39A+CDH2-OE组(转染shDDX39A慢病毒和过表达CDH2病毒)。利用Celigo法、MTT法和Transwell实验分别检测过表达CDH2对DDX39A敲减细胞增殖能力、活力以及转移能力的影响。结果与正常组织相比,食管鳞癌组织中DDX39A的表达显著上调(P<0.001)。DDX39A及其相关基因主要作用于遗传信息的翻译与加工和细胞凋亡等过程。与shCtrl组相比,shDDX39A组KYSE-150和TE-1细胞中DDX39A的mRNA和蛋白表达显著降低(P<0.01),KYSE-150和TE-1细胞增殖和细胞活力显著下降(P<0.001),KYSE-150和TE-1细胞的克隆形成能力显著降低(P<0.01),KYSE-150和TE-1细胞的凋亡水平显著升高(P<0.01),KYSE-150和TE-1细胞的迁移和侵袭能力显著下降(P<0.001)。裸鼠成瘤实验提示DDX39A敲减后瘤体的体积和质量均明显减少(P<0.001)。与shDDX39A+NC-OE组相比,shDDX39A+CDH2-OE组KYSE-150细胞的增殖、细胞活力以及转移能力均显著提高(P<0.001)。结论DDX39A可作为食管鳞癌诊治的新靶点,上调其表达可能通过诱导上皮间充质转化促进食管鳞癌细胞生长、迁移与侵袭。

Objective To explore the effect of DDX helicase 39A(DDX39A)on the biological behaviors of esophageal squamous cell carcinoma(ESCC)KYSE-150 and TE-1 cells and its related mechanism.Methods Differential expression of DDX39A in ESCC tumor tissues and normal tissues was analyzed using TCGA and GEO databases.Spearman correlation analysis was performed in TCGA database to obtain the most related genes for DDX39A expression(r>0.5 and P<0.05).The biological function of DDX39A in ESCC was annotated by KEGG and GSEA enrichment analysis.The expression of DDX39A in KYSE-150 and TE-1 cells was downregulated by lentiviral-mediated short hairpin RNA(shRNA)interfering technology,and then the cells were divided into shCtrl group and shDDX39A group.The knockdown efficiency was detected by qPCR and Western blot.Celigo method,colony formation and MTT assays were performed to test the proliferative capacity and the viability of KYSE-150 and TE-1 cells.Flow cytometry was used to detect the apoptosis of KYSE-150 and TE-1 cells.Migration and invasion capabilities of KYSE-150 and TE-1 cells were measured through Transwell assay.The effect of knocking down DDX39A on tumors in vivo was detected by tumor formation experiment in nude mice.Western blot was used to screen the tumor-associated classical pathway molecules in KYSE-150 cells with significant changes in the levels of intracellular protein expression after knocking down DDX39A.The KYSE-150 cell lines with stable screened molecule CDH2(Cadherin 2)overexpression were constructed by lentiviral transfection,and the cells were divided into shDDX39A+NC-OE group(transfected with shDDX39A lentivirus and virus overexpressing empty control vector)and shDDX39A+CDH2-OE group(transfected with shDDX39A lentivirus and virus overexpressing CDH2 vector).Celigo method,MTT assay and Transwell assay were adopted to detect the proliferative capacity,the viability and the metastasis of DDX39A knockdown cells after overexpressing CDH2.Results The expression of DDX39A was significantly upregulated in ESCC tissues compared to normal tissues(P<0.001).DDX39A and its related genes mainly functioned in processes such as translation and processing of genetic information and cell apoptosis.Compared with shCtrl group,the mRNA and protein expression levels of DDX39A were significantly reduced in shDDX39A group(P<0.01),the proliferation and the viabilities of KYSE-150 and TE-1 cells were significantly decreased(P<0.001),the clone-forming abilities of KYSE-150 and TE-1 cells were significantly reduced(P<0.01),the apoptosis of KYSE-150 and TE-1 cells was significantly increased(P<0.01),and the migration and invasion capabilities of KYSE-150 and TE-1 cells were significantly decreased(P<0.001).Tumor formation experiments in nude mice suggested that the volume and the weight of tumors in DDX39A knockdown group were significantly reduced compared with shCtrl group(P<0.001).Compared with shDDX39A+NC-OE group,the proliferation,the viability and the metastatic capacity of KYSE-150 cells were significantly increased in shDDX39A+CDH2-OE group(P<0.001).Conclusion DDX39A can serve as a novel target for the diagnosis and treatment of ESCC.DDX39A overexpression may promote the growth,migration and invasion of ESCC cells by inducing EMT.