下调TRAF6表达对肺癌细胞株恶性生物学行为的影响

摘要

Background and objectiveIt has been proven that tumor necrosis factor receptor-associated factor 6 (TARF6) was a commonly ampliifed oncogene in lung cancer. However, the precise role of TARF6 protein in lung cancer has not been extensively investigated. hTis study analyzed the effects of TARF6 on the proliferation, apoptosis, cell cycle, migration, and invasion capability of lung cancer cell lines, as well as the potential molecular mechanisms involved.MethodsTo address the expression of TARF6 in lung cancer cells, four lung cancer cell lines (A549, H1650, SPC-A-1 and Calu-3) were assayed to determine the expression of TARF6 protein by Western blot and TARF6 mRNA via qRT-PCR. Moreover, siRNA targeting TARF6 was introduced into SPC-A-1 and Calu-3 cells. Nuclear factor-қB (NF-қB) DNA-binding activity, apoptosis rates, cell proliferation, cell cycle, migration, and invasion were determined by electrophoretic mobility shitf assay, lfow cytometry, MTS assay, lfow cytometry, scratch test, and transwell chamber assay, respectively. Western blot analysis was also performed to evalu-ate the expression of the following proteins through K63-ubiquitination: P65, CD24 and CXCR4. Whole-genome sequencing analysis was conducted using a second-generation sequencer in SPC-A-1 cells.Results TARF6 was highly up-expressed in SPC-A-1 and Calu-3 cell lines than the other two cells, which also showed K63-ubiquitinization in TARF6. However, consti-tutive activation of NF-қB was observed only in SPC-A-1 lung cancer cells. Downregulation of TARF6 suppressed the NF-κB activation, cell migration, and invasion but promoted the cell apoptosis of SPC-A-1 cells. Markedly decreased expression of CD24 and CXCR4 was observed in SPC-A-1 cells transfected by TARF6 siRNA. Nevertheless, TARF6 downregulation did not affect the proliferation and cell cycle of SPC-A-1 cells. Additionally, TARF6 regulation did not affect the proliferation, apoptosis, cell cycle, migration, and invasion of Calu-3 cells. No mutations and no changes in gene copy numbers of TARF6 were found by whole-exome sequencing of SPC-A-1 cells.ConclusionTARF6 may be involved in cell migration, invasion, and apoptosis of SPC-A-1 cells, possibly through regulating the NF-қB-CD24/CXCR4 pathway.%背景与目的已有的研究提示肿瘤坏死因子受体相关因子6（tumor necrosis factor receptor-associated factor 6, TRAF6）在肺癌中常常扩增，可能扮演癌基因角色，但TRAF6的确切作用尚未充分阐明。本研究探索TRAF6表达对肺癌细胞株的增殖、凋亡、细胞周期、迁移及侵袭能力的影响以及可能作用机制。方法选用A549、H1650、SPC-A-1以及Calu-3等四种肺癌细胞株，应用蛋白印迹、qRT-PCR检测其TARF6蛋白及mRNA表达。SPC-A-1、Calu-3细胞转染TRAF6 siRNA，以EMSA方法检测不同处理组核因子-κB的DNA结合活性， MTS法检测细胞增殖，流式细胞仪PI染色检测细胞凋亡，流式细胞仪进行细胞周期测定，划痕实验及Tran-swell小室法检测细胞迁移及侵袭能力，并应用蛋白印迹检测泛素化抗体、p65、CD24、CXCR4等蛋白表达。SPC-A-1细胞提取DNA后，应用二代测序法进行全基因组测序。结果在四种细胞株中，SPC-A-1和Calu-3细胞TRAF6相对高表达，TRAF6发生自身K63-泛素化，但仅在SPC-A-1细胞中观察到核因子-κB组成性活化。转染TRAF6 siRNA后，SPC-A-1、Calu-3细胞TRAF6表达明显下调，与空白组及对照组相比，下调TRAF6表达可抑制SPC-A-1细胞核因子-κB活性、降低迁移及侵袭能力以及促进细胞凋亡，CD24和CXCR4的表达也明显下调，但对细胞增殖及细胞周期无明显影响。下调TARF6表达对Calu-3细胞株的核因子-κB活性、细胞增殖、凋亡、细胞周期、迁移及侵袭能力等均无明显影响。未发现SPC-A-1细胞株TRAF6基因突变或拷贝数改变。结论下调TRAF6表达可抑制SPC-A-1细胞迁移及侵袭能力，促进细胞凋亡，并且TRAF6可能是通过调控核因子-κB-CD24/CXCR4信号通路参与调控肺癌侵袭、细胞凋亡。