SEZ6L2 Regulates the Proliferation and Apoptosis of Lung Squamous Cell Carcinoma Cells through ECM-Receptor Signaling
WU Feng1, LIU Chuanping2, GAO Guogang2, YU Ge2*
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.
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