转染IκBα基因抑制NF-κB活性对A549细胞侵袭行为的影响

Inhibition of NF-κΒ Through IκBα Transfection Affects Invasion of Human Lung Cancer Cell Line A549

背景与目的:近年来的研究显示核因子-κB(nuclear factor-κB,NF-κB)的活化能够调节肿瘤细胞的侵袭和转移。本研究探讨抑制NF-κB的活性对A549细胞侵袭能力的影响及其可能的机制。方法:构建表达NF-κB抑制物α同分异构体(inhibitor of NF-κB,α isoform,IκBα)的真核表达重组质粒pcDNA3.1(+)/IκBα。体外培养A549细胞,分为未转染组(不转染质粒)、转染pcDNA3.1(+)组、转染pcDNA3.1(+)/IκBα组,分别转染相应的质粒。应用RT-PCR、Westernblot检测各组细胞IκBα的表达情况,应用凝胶电泳迁移率改变实验(electrophoretic mobility shift assay,EMSA)检测各组细胞NF-κB的活性,应用Transwell侵袭小室检测各组细胞的侵袭能力,应用RT-PCR方法检测各组细胞MMP-2、MMP-9的mRNA水平,应用明胶酶谱法检测各组细胞基质金属蛋白酶-2(matrix metalloproteinase-2,MMP-2)、基质金属蛋白酶-9(matrix metalloproteinase-9,MMP-9)的活性。结果:酶切鉴定结果显示pcDNA3.1(+)/IκBα质粒构建成功。RT-PCR和Westernblot检测结果表明构建的pcDNA3.1(+)/IκBα质粒能够在A549细胞中表达IκBα。转染pcDNA3.1(+)/IκBα组A549细胞NF-κΒ活性、侵袭细胞数目、MMP-2活性及MMP-9活性均低于未转染组和转染pcDNA3.1(+)组(P值均<0.05),而转染pcDNA3.1(+)组与未转染组之间的差异均无统计学意义(P值均>0.05)。结论:转染IκBα基因能够抑制A549细胞中NF-κB的活性。NF-κB的活性下降能够抑制A549细胞的侵袭能力,其机制可能与MMP-2、MMP-9表达下调有关。

BACKGROUND ＆ OBJECTIVE. Recent studies have shown that activation of nuclear factor-KB （NF-KB） can regulate the invasion and metastasis of cancer cells. The present study was to investigate inhibition of NF-KB activity on invasion of human lung cancer cell line A549 and the possible mechanism. METHODS： The recombinant plasmid pcDNA3.1 （＋）/ IkBα, expressing the a isoform （IKBa） of the NF-KB inhibitor, was constructed. A549 cells were cultured in vitro and divided into nontransfection group, pcDNA3.1 （＋） transfected group and pcDNA3.1 （＋）/IKBa transfected group. The expression of IkBα was detected by reverse transcription-polymerase chain reaction （RT-PCR） and Western blotting. The activity of NF-KB was determined by electrophoretic mobility shift assay （EMSA）. Invasion of A549 cells was assessed by transwell chamber assay. The expression of MMP-2 and MMP-9 was detected by RT-PCR and gelatin zymography. RESULTS. Plasmid pcDNA3.1 （＋）/IKBa was successfully constructed and expressed in A549 cells. The activity of NF-KB, the number of invasive cells, the activity of MMP-2 and MMP-9 of A549 cells in pcDNA3.1 （＋）/IkBα transfected group were significantly lower than those in non-transfection group and pcDNA3.1 （＋） transfected group （all P〈0.05）. CONCLUSION： Transfection of IkBα can inhibit NF-KB activity, thus inhibit cell invasion of A549, which may be through the down-regulation of MMP-2 and MMP-9 expressions.