ANKRD49对人肺腺癌细胞迁移能力的影响及其机制

Effect of ANKRD49 on migration of human lung adenocarcinoma cells and its mechanism

目的 研究ANKRD49(ankyrin repeat domain 49)对人肺腺癌细胞NCI-H1299迁移能力的影响及其机制。方法将携带ANKRD49基因的慢病毒载体及针对ANKRD49的shRNA感染NCI-H1299细胞,构建稳定过表达与敲低ANKRD49的细胞模型,同时分别构建感染空白序列慢病毒载体和无意义shRNA序列慢病毒载体的对照细胞模型。采用实时荧光定量PCR和Western blot法检测ANKRD49 mRNA和蛋白表达水平;细胞划痕试验检测ANKRD49对细胞迁移能力的影响;实时荧光定量PCR和Western blot法检测细胞基质金属蛋白酶(matrix metalloproteinase,MMP)-2/9及基质金属蛋白酶组织抑制因子(tissue inhibitor of metalloprotease,TIMP)-1/2 mRNA及蛋白表达水平;Western blot法检测p65、p-p65、IκBα和p-IκBα蛋白表达水平。结果 ANKRD49过表达组细胞中ANKRD49 mRNA和蛋白水平均显著高于对照组(t分别为70.02和45.68,P均<0.001)。与对照组相比,ANKRD49过表达组细胞24和48 h的迁移能力明显升高(t分别为5.343和3.282,P分别为0.005 9和0.030 4);MMP-2和MMP-9 mRNA转录水平及蛋白表达水平均明显升高(t分别为9.304和6.193,P分别为0.000 7和0.003 5),TIMP-1和TIMP-2 mRNA转录水平及蛋白表达水平均明显下降(t分别为3.858和3.517,P分别为0.018 2和0.024 5),MMP-2/TIMP-1和MMP-9/TIMP-2值明显升高(t分别为17.7和9.682,P分别<0.001和<0.01);p-p65和p-IκBα蛋白表达水平明显升高,p65和IκBα总蛋白水平无明显变化,p-p65/p65、p-IκBα/IκBα值明显增高(t分别为3.962和5.370,P分别为0.016 7和0.005 8)。ANKRD49敲低后结果均与过表达结果相反。结论 ANKRD49通过激活NF-κB/p65信号通路增加MMP-2/9的表达及提高MMP-9/TIMP-1、MMP-2/TIMP-2值来促进NCI-H1299细胞的迁移。

Objective To study the effect of ankyrin repeat domain 49(ANKRD49)on the migration of human lung adenocarcinoma cell line NCI-H1299 and its mechanism.Methods NCI-H1299 cells were infected with lentivirus vector carrying ANKRD49 gene and shRNA targeting ANKRD49 to construct the cell models stably overexpressing and knocking down ANKRD49.Meanwhile,the control cell models infected with empty lentivirus vector and lentivirus vector with scramble sequences were constructed respectively.The expression levels of ANKRD49 mRNA and protein were detected by real-time fluorescence quantitative PCR and Western blot.The effect of ANKRD49 on cell migration was measured by scratch test.The mRNA and protein levels of matrix metalloproteinase(MMP)-2/9 and tissue inhibitor of metalloproteinase(TIMP)-1/2 were detected by real-time fluorescence quantitative PCR and Western blot.The protein expression levels of p65,p-p65,IκBα and p-IκBα were detected by Western blot.Results The levels of ANKRD49 mRNA and protein in the ANKRD49 overexpression group were significantly higher than those in the control group(t = 70.02 and 45.68,respectively,each P < 0.001).Compared with the control group,the migration ability of cells in the ANKRD49 overexpression group significantly increased at 24 h and 48 h(t = 5.343 and 3.282,P = 0.005 9 and 0.030 4,respectively);The mRNA transcription levels and protein expression levels of MMP-2 and MMP-9 significantly increased(t = 9.304 and 6.193,P =0.000 7 and 0.003 5,respectively),while the mRNA and protein expression of TIMP-1 and TIMP-2 decreased significantly(t = 3.858 and 3.517,P = 0.018 2 and 0.024 5,respectively),and the values of MMP-2/TIMP-1 and MMP-9/TIMP-2 significantly increased(t = 17.7 and 9.682,P < 0.001 and < 0.01,respectively);The expression of p-p65 and pIκBα significantly increased,the total protein levels of p65 and IκBα showed no obvious change,and the values of p-p65/p65 and p-IκBα/IκBα significantly increased(t = 3.962 and 5.370,P = 0.016 7 and 0.005 8,respectively).However,knocking down of ANKRD49 presented the opposite results.Conclusion ANKRD49 promotes the migration of NCI-H1299cells by enhan-cing the expression of MMP-2/9,the values of MMP-9/TIMP-1 and MMP-2/TIMP-2 via activating NF-κB/p65 signa-ling pathway.