沉默DLX4基因表达可抑制前列腺癌细胞的迁移和侵袭

Silencing DLX4 gene expression inhibits the migration and invasion of prostate cancer cells

目的:探讨抑制DLX4(distal-less homeobox 4)基因的表达对人前列腺癌细胞增殖、迁移和侵袭的影响,以及其可能的作用机制。方法:分别采用实时荧光定量PCR和蛋白质印迹法检测人前列腺癌LNCap、DU145和PC-3细胞中DLX4 mRNA和蛋白的表达水平。将DLX4 shRNA重组慢病毒pLKO.1-puro-shDLX4感染PC-3细胞(称为PC-3-shDLX4细胞)后,应用实时荧光定量PCR和蛋白质印迹法分别验证DLX4的敲除效果,应用MTT法和Transwell小室法分别检测PC-3-shDLX4细胞的增殖、迁移和侵袭能力,应用蛋白质印迹法检测PC-3-shDLX4细胞中上皮-间质转化(epithelial-mesenchymal transition,EMT)相关标志物E-钙黏蛋白(E-cadherin)和波形蛋白(Vimentin)以及参与调控EMT的重要转录因子Snail和Twist蛋白的表达水平。结果:DLX4 mRNA和蛋白在人前列腺癌LNCap、DU145和PC-3细胞中均有较高水平的表达,PC-3细胞中DLX4 mRNA和蛋白的表达水平均高于LNCap和DU145细胞(P值均<0.05)。重组慢病毒pLKO.1-puroshDLX4感染PC-3细胞后,PC-3细胞中DLX4基因表达被有效沉默,PC-3-shDLX4细胞中DLX4 mRNA和蛋白的表达水平均明显下调(P值均<0.05)。PC-3-shDLX4细胞的增殖能力未发生明显改变(P>0.05),而其迁移及侵袭能力却明显下降(P值均<0.05)。PC-3-shDLX4细胞中Vimentin蛋白的表达水平明显下调(P<0.05),而E-cadherin蛋白的表达水平明显上调(P<0.05);参与调控EMT的Snail和Twist蛋白表达水平在PC-3-shDLX4细胞中亦明显下调(P值均<0.05)。结论:DLX4在前列腺癌细胞中表达水平较高,下调DLX4表达可能通过逆转Snail和Twist介导的EMT来抑制前列腺癌PC-3细胞的迁移和侵袭。

Objective： To investigate the effects of inhibiting the expression ofdistal-less homeobox 4（DLX4） gene on the proliferation, migration and invasion of human prostate cancer cells, and to explore its possible mechanism.Methods： The expression levels of DLX4 mRNA and protein in human prostate cancer LNCap,DU145 and PC-3 cells were detected by real-time fluorescent quantitative PCR and Western blotting, respectively. After the DLX4 shRNA recombination lentiviral vector pLKO.1-puroshDLX4 was transfected into PC-3 cells（named as PC-3-shDLX4 cells）, the efficiency of DLX4 knockdown was verified by real-time fluorescent quantitative PCR and Western blotting,respectively. The proliferation, migration and invasion of PC-3-shDLX4 cells were measured by MTT method and Transwell assay, respectively. The expression levels of epithelialmesenchymal transition（EMT） markers including E-cadherin and Vimentin, as well as the key transcription factors to regulate EMT including Snail and Twist were detected by Western blotting.Results： DLX4 mRNA and protein were highly expressed in prostate cancer LNCap, DU145 and PC-3 cells. The expression levels of DLX4 mRNA and protein in PC-3 cells were higher than those in LNCap and DU145 cells（all P 〈0.05）. The expression of DLX4 gene in PC-3 cells after transfection with the recombination lentiviral vector pLKO.1-puro-shDLX4 was effectively silenced. The expression levels of DLX4 mRNA and protein in PC-3-shDLX4 cells were down-regulated（both P 〈0.05）. The proliferation ability of PC-3-shDLX4 cells was almost undisturbed（P 〉0.05）, and the migration and invasion abilities were decreased（both P 〈0.05）. The expression level of Vimentin in PC-3-shDLX4 cells was down-regulated（P 〈0.05）, and the expression level of E-cadherin in PC-3-shDLX4 cells was up-regulated（P 〈0.05）. Furthermore, the expression levels of Snail and Twist involved in EMT regulation in PC-3-shDLX4 cells were down-regulated（both P〈 0.05）.Conclusion： DLX4 is highly expressed in prostate cancer cells. Depletion of DLX4 gene may inhibit the migration and invasion of PC-3 cells by counteracting EMT mediated by Snail and Twist.