中国肺癌杂志
中國肺癌雜誌
중국폐암잡지
Chinese Journal of Lung Cancer
2015年
11期
661-667
,共7页
林根%黄传钟%苏光建%胡卉华%徐海鹏%黄诚
林根%黃傳鐘%囌光建%鬍卉華%徐海鵬%黃誠
림근%황전종%소광건%호훼화%서해붕%황성
肺肿瘤%肿瘤坏死因子受体相关因子6%核因子-κB%凋亡%侵袭
肺腫瘤%腫瘤壞死因子受體相關因子6%覈因子-κB%凋亡%侵襲
폐종류%종류배사인자수체상관인자6%핵인자-κB%조망%침습
Lung neoplasms%Tumor necrosis factor receptor-associated factor 6%Nuclear factor-kappa B%Apopto-sis%Invasion
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。
揹景與目的已有的研究提示腫瘤壞死因子受體相關因子6（tumor necrosis factor receptor-associated factor 6, TRAF6）在肺癌中常常擴增，可能扮縯癌基因角色，但TRAF6的確切作用尚未充分闡明。本研究探索TRAF6錶達對肺癌細胞株的增殖、凋亡、細胞週期、遷移及侵襲能力的影響以及可能作用機製。方法選用A549、H1650、SPC-A-1以及Calu-3等四種肺癌細胞株，應用蛋白印跡、qRT-PCR檢測其TARF6蛋白及mRNA錶達。SPC-A-1、Calu-3細胞轉染TRAF6 siRNA，以EMSA方法檢測不同處理組覈因子-κB的DNA結閤活性， MTS法檢測細胞增殖，流式細胞儀PI染色檢測細胞凋亡，流式細胞儀進行細胞週期測定，劃痕實驗及Tran-swell小室法檢測細胞遷移及侵襲能力，併應用蛋白印跡檢測汎素化抗體、p65、CD24、CXCR4等蛋白錶達。SPC-A-1細胞提取DNA後，應用二代測序法進行全基因組測序。結果在四種細胞株中，SPC-A-1和Calu-3細胞TRAF6相對高錶達，TRAF6髮生自身K63-汎素化，但僅在SPC-A-1細胞中觀察到覈因子-κB組成性活化。轉染TRAF6 siRNA後，SPC-A-1、Calu-3細胞TRAF6錶達明顯下調，與空白組及對照組相比，下調TRAF6錶達可抑製SPC-A-1細胞覈因子-κB活性、降低遷移及侵襲能力以及促進細胞凋亡，CD24和CXCR4的錶達也明顯下調，但對細胞增殖及細胞週期無明顯影響。下調TARF6錶達對Calu-3細胞株的覈因子-κB活性、細胞增殖、凋亡、細胞週期、遷移及侵襲能力等均無明顯影響。未髮現SPC-A-1細胞株TRAF6基因突變或拷貝數改變。結論下調TRAF6錶達可抑製SPC-A-1細胞遷移及侵襲能力，促進細胞凋亡，併且TRAF6可能是通過調控覈因子-κB-CD24/CXCR4信號通路參與調控肺癌侵襲、細胞凋亡。
배경여목적이유적연구제시종류배사인자수체상관인자6（tumor necrosis factor receptor-associated factor 6, TRAF6）재폐암중상상확증，가능분연암기인각색，단TRAF6적학절작용상미충분천명。본연구탐색TRAF6표체대폐암세포주적증식、조망、세포주기、천이급침습능력적영향이급가능작용궤제。방법선용A549、H1650、SPC-A-1이급Calu-3등사충폐암세포주，응용단백인적、qRT-PCR검측기TARF6단백급mRNA표체。SPC-A-1、Calu-3세포전염TRAF6 siRNA，이EMSA방법검측불동처리조핵인자-κB적DNA결합활성， MTS법검측세포증식，류식세포의PI염색검측세포조망，류식세포의진행세포주기측정，화흔실험급Tran-swell소실법검측세포천이급침습능력，병응용단백인적검측범소화항체、p65、CD24、CXCR4등단백표체。SPC-A-1세포제취DNA후，응용이대측서법진행전기인조측서。결과재사충세포주중，SPC-A-1화Calu-3세포TRAF6상대고표체，TRAF6발생자신K63-범소화，단부재SPC-A-1세포중관찰도핵인자-κB조성성활화。전염TRAF6 siRNA후，SPC-A-1、Calu-3세포TRAF6표체명현하조，여공백조급대조조상비，하조TRAF6표체가억제SPC-A-1세포핵인자-κB활성、강저천이급침습능력이급촉진세포조망，CD24화CXCR4적표체야명현하조，단대세포증식급세포주기무명현영향。하조TARF6표체대Calu-3세포주적핵인자-κB활성、세포증식、조망、세포주기、천이급침습능력등균무명현영향。미발현SPC-A-1세포주TRAF6기인돌변혹고패수개변。결론하조TRAF6표체가억제SPC-A-1세포천이급침습능력，촉진세포조망，병차TRAF6가능시통과조공핵인자-κB-CD24/CXCR4신호통로삼여조공폐암침습、세포조망。
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-?B (NF-?B) 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-?B 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-?B-CD24/CXCR4 pathway.
