SEZ6L2通过ECM-受体信号调节肺鳞癌细胞的增殖与凋亡

SEZ6L2 Regulates the Proliferation and Apoptosis of Lung Squamous Cell Carcinoma Cells through ECM-Receptor Signaling

该研究探讨癫痫相关6同源物样2(seizure-related 6 homolog like 2,SEZ6L2)对肺鳞癌(lung squamous cell carcinoma,LUSC)细胞的增殖与凋亡的影响及其潜在的作用机制。该研究采用癌症基因组图谱数据库(The Cancer Genome Atlas,TCGA)分析SEZ6L2在肺鳞癌组织中的表达情况;采用实时逆转录聚合酶链式反应(real-time reverse transcriptase-polymerase chain reaction,RTqPCR)和蛋白质印迹法(Westernblot)检测正常人支气管上皮细胞BEAS-2B和肺鳞癌细胞(NCIH520、NCI-H1703、SK-MES-1)中SEZ6L2表达情况;采用RT-qPCR和Western blot检测SEZ6L2的干扰效率;采用CCK-8、EdU染色法和细胞克隆实验检测细胞增殖水平;采用细胞划痕和侵袭实验检测细胞迁移水平和侵袭能力;采用流式细胞术和Western blot检测细胞凋亡水平及凋亡相关蛋白的表达水平;借助基因集合富集分析(gene set enrichment analysis,GSEA)软件,分析SEZ6L2在细胞外基质(extracellular matrix,ECM)-受体信号中的富集情况;借助linkedomics和基于基因表达水平值的交互式分析平台(gene expression profiling interactive analysis,GEPIA)分析SEZ6L2与ECM-受体信号蛋白整合素Β3(integrin beta 3,ITGB3)、整合素Β6(integrin beta 6,ITGB6)、整合素α3(integrin alpha3,ITGA3)的关系;采用Western blot检测ITGB3、ITGB6、ITGA3及其下游信号蛋白磷酸化黏着斑激酶(phosphorylated focal adhesion kinase,p-FAK)、p-SRC、FAK、SRC的表达情况。该研究发现,TCGA数据库显示SEZ6L2在肺鳞癌组织中的表达水平增加,且表达水平越高LUSC患者预后越差;与BEAS-2B组比较,SEZ6L2表达在肺鳞癌细胞NCI-H520、NCI-H1703和SK-MES-1中显著上调,且在NCI-H1703细胞中表达水平最高。因此,选择NCI-H1703细胞做后续研究。构建SEZ6L2的干扰质粒,SEZ6L2在si-SEZ6L2-3组中表达水平更低,表现出更好的转染效率,因而选择si-SEZ6L2-3(以下简称为si-SEZ6L2)进行后续研究。干扰SEZ6L2表达后,与si-NC组比较NCI-H1703细胞增殖、迁移和侵袭水平下降,凋亡水平上升,促凋亡蛋白Bax和cleaved caspase3表达水平增加,抗凋亡蛋白BCL2表达水平下降,ECM-受体信号蛋白ITGB3、ITGB6、ITGA3及其下游信号蛋白p-FAK、p-SRC表达水平下调。该研究得出,SEZ6L2通过ECM-受体信号调节肺鳞癌细胞的增殖、迁移、侵袭和凋亡。

This study was designed to explore the role of SEZ6L2(seizure-related 6 homolog like 2)in the proliferation and apoptosis of LUSC(lung squamous cell carcinoma)cells as well as to investigate the hidden mechanism.This study used TCGA(The Cancer Genome Atlas)database to analyze the expression of SEZ6L2 in LUSC tissues.The expressions of SEZ6L2 in normal human bronchial epithelial cells BEAS-2B and LUSC cell lines NCI-H520,NCI-H1703 and SK-MES-1 were detected using RT-qPCR(real-time reverse transcriptase-polymerase chain reaction)and Western blot.Following the construction of SEZ6L2 interference plasmids,cells were divided into Control group,si-NC group and si-SEZ6L2-1/2 group.The transfection efficiency of si-SEZ6L2 was examined with RT-qPCR and Western blot.The cell proliferation was detected using CCK-8(cell counting kit-8)assay,EdU(5-ethynyl-2’-deoxyuridine)staining and colony formation assay.Cell migration and invasion were detected using wound healing and transwell assay.Cell apoptosis and apoptosis-related proteins were detected using flow cytometry and Western blot.With GSEA(gene set enrichment analysis)database,the enrichment of SEZ6L2 in ECM(extracellular matrix)-receptor signaling was detected.With linkedomics and GEPIA(gene expression profiling interactive analysis)databases,the relationship between SEZ6L2 and ECM-receptor signaling proteins ITGB3(integrin beta 3),ITGB6I(integrin beta 6)and ITGA3(integrin alpha 3)was detected.The expressions of ITGB3,ITGB6,ITGA3 and the downstream signaling proteins p-FAK(phosphorylated focal adhesion kinase),p-SRC,FAK and SRC were detected using Western blot.The present study found that according to TCGA database,SEZ6L2 expression was upregulated in LUSC tissues and SEZ6L2 upregulation indicated poorer prognosis of LUSC patients.Compared with the BEAS-2B group,SEZ6L2 expression was significantly increased in LUSC cells NCI-H520,NCI-H1703 and SK-MES-1.SEZ6L2 had the highest expression in NCI-H1703 cells and thus NCI-H1703 cells were chosen for following experiments.Following the construction of SEZ6L2 interfering plasmids,it was found that SEZ6L2A had the lowest expression in si-SEZ6L2-3 group.Therefore,si-SEZ6L2-3(hereinafter referred as siSEZ6L2)was selected for following experiments.After interfering SEZ6L2 expression,NCI-H1703 cell proliferation,migration and invasion were decreased,cell apoptosis was increased,the expressions of pro-apoptotic proteins Bax and cleaved caspase3 were increased,anti-apoptotic protein BCL2 expression was increased,the expressions of ECM-receptor signaling proteins ITGB3,ITGB6,ITGA3 and the downstream signaling proteins p-FAK and p-SRC were decreased when compared with the si-NC group.It can be concluded that SEZ6L2 regulates the proliferation,migration,invasion and apoptosis of LUSC cells through ECM-receptor signaling.