PAX9基因在肺鳞状细胞癌中的表达及其临床意义

Expression of PAX9 gene in lung squamous cell carcinoma and its clinical significance

目的探讨PAX9基因在肺鳞状细胞癌(LUSC)中的表达情况及其临床意义。方法从TCGA数据库和GEO数据库中获取LUSC患者的基因测序数据和预后数据,包括TCGA-LUSC数据集、GSE21933数据集和GSE73403数据集;选取34例LUSC患者的癌组织及癌旁组织。基于TCGA-LUSC数据集和GSE21933数据集绘制ROC曲线,评估PAX9诊断LUSC的效能;建立基于TCGA-LUSC数据集和GSE73403数据集的LUSC患者预后列线图模型,并采用Kaplan-Meier法分析不同PAX9表达水平LUSC患者的总体生存率;采用qRT-PCR检测比较LUSC患者癌组织与癌旁组织的PAX9基因表达水平;采用qRT-PCR、蛋白免疫印迹试验(Western Blot)检测比较PAX9在人肺鳞状细胞癌细胞(NCI-H520)和正常肺泡上皮细胞(BEAS-2B)中的相对表达情况。用siRNA实现PAX9的敲低,并通过CCK8试验、划痕试验、平板克隆试验以及细胞周期试验分析PAX9的下调对LUSC细胞活力、迁移、扩增以及细胞周期的影响。结果基于TCGA-LUSC数据集和GSE21933数据集,绘制ROC曲线的分析结果显示,根据PAX9基因表达水平诊断LUSC的效能良好;生存分析结果显示,PAX9低表达LUSC患者的总体生存率显著低于PAX9高表达的LUSC患者(P<0.05)。LUSC患者癌组织PAX9基因表达水平显著低于癌旁组织;NCI-H520 PAX9基因和蛋白的表达水平均显著低于BEAS-2B。PAX9基因敲除试验结果显示,si-PAX9转染组的NCI-H520细胞活力显著高于si-NC转染组,克隆数显著多于si-NC转染组,愈合速度显著快于si-NC转染组;与si-NC转染组比较,si-PAX9转染组NCI-H520细胞处于S期的细胞明显减少,处于G2期的细胞明显增加。结论PAX9在LUSC中的表达水平可作为LUSC患者诊断和预后的评估指标;敲除PAX9基因会显著影响LUSC细胞的增殖、克隆、迁移和细胞周期进程。

Objective To investigate the expression of PAX9 gene in lung squamous cell carcinoma(LUSC)and its clinical significance.Methods The gene sequencing data and prognostic data of LUSC patients were obtained from the TCGA database and GEO database,including the data set of TCGA-LUSC,GSE21933 and GSE73403.Cancerous tissues and tissues adjacent to cancer from 34 LUSC patients were selected.The ROC curve was drawn based on the data set of TCGA-LUSC and GSE21933 to evaluate the efficacy of PAX9 in diagnosing LUSC.A prognostic nomogram model of LUSC patients based on the data set of TCGA-LUSC and GSE73403 was established,and the overall survival rate of LUSC patients with different PAX9 expressions was analyzed by the Kaplan-Meier method.qRT-PCR was used to detect and compare the expression levels of PAX9 gene between cancerous tissues and tissues adjacent to cancer in LUSC patients.qRT-PCR and Western Blot were used to detect and compare relative expressions of PAX9 between human lung squamous cell carcinoma(NCI-H520)and normal alveolar epithelial cells(BEAS-2B).siRNA was used to achieve the knockdown of PAX9,and the effects of PAX9 down-regulation on LUSC cell viability,migration,amplification and cell cycle were analyzed by CCK8 test,scratch test,plate cloning test and cell cycle test.Results Based on the data set of TCGA-LUSC and GSE21933,the ROC curve analysis results showed that the efficacy of PAX9 gene expression in diagnosing LUSC was good.The survival analysis results showed that the overall survival rate of LUSC patients with PAX9 low expression was significantly lower than that of LUSC patients with PAX9 high expression(P<0.05).The expression level of PAX9 gene in cancerous tissues of LUSC patients was significantly lower than that in tissues adjacent to cancer.PAX9 gene and protein expression levels in NCI-H520 were significantly lower than those in BEAS-2B.The results of PAX9 gene knockout test revealed that,compared with the si-NC transfection group,NCI-H520 cells in the si-PAX9 transfection group yielded higher viability,higher number of clones,and faster healing speed;meanwhile,the number of NCI-H520 cells in the S phase of the si-PAX9 transfection group significantly reduced,and the number of cells in the G2 phase significantly increased.Conclusion The expression level of PAX9 can be used as an indicator for the diagnosis and prognosis of LUSC patients.Knocking out the PAX9 gene will prominently affect the proliferation,cloning,migration and cell cycle progression of LUSC cells.