慢病毒介导的TPX2沉默对人宫颈癌HeLa细胞凋亡和侵袭的影响及机制

The Effect of Silencing TPX2 Gene Expression Mediated by Lentiviral on Apoptosis and Invasion in Human Cervical Cancer HeLa Cells and Its Mechanism

该文的目的是研究慢病毒介导的Xklp2靶蛋白(targeting protein for Xklp2,TPX2)沉默对人宫颈癌He La细胞凋亡、侵袭的影响及机制。构建4种载有TPX2-sh RNA的重组慢病毒及其阴性对照,将5种重组慢病毒分别稳定感染人宫颈癌He La细胞,利用实时荧光定量PCR和Western blot筛选出TPX2沉默效果最佳的一组He La细胞作为干扰组进行后续实验。采用Transwell基底膜侵袭实验测定各组细胞的侵袭能力。采用流式细胞术检测各组细胞的凋亡情况。Western blot检测TPX2-sh RNA转染前后细胞凋亡及侵袭相关蛋白质Bcl-2、Bax、Caspase-3、MMP9、TIMP-1及nm23-H1水平。结果显示,筛选出的RNA干扰慢病毒载体LV-TPX2-sh RNA-1可有效抑制He La细胞TPX2的表达;与对照组相比,干扰组的He La细胞凋亡率明显增加(P<0.01);穿过Transwell小室基底膜细胞明显减少(P<0.01)。He La细胞感染LV-TPX2-sh RNA-1能下调凋亡相关蛋白Bcl-2的表达水平,上调Caspase-3及Bax的表达水平(P<0.05);上调侵袭相关蛋白质nm23-H1及TIMP-1水平,下调MMP9水平(P<0.05)。以上结果表明,沉默TPX2表达能增加宫颈癌细胞的凋亡,可能与其上调Caspase-3及Bax水平,下调Bcl-2水平有关;沉默TPX2表达能抑制宫颈癌细胞侵袭能力,可能与其上调TIMP-1及nm23-H1水平,下调MMP9的水平有关。

This article was aimed to investigate the effect of silencing TPX2 expression mediated by lentiviral on apoptosis and invasion in human cervical cancer HeLa cells and its mechanism. The four lentivirus expression vectors carried TPX2-shRNA and the negative control were constructed. The recombinant lentivirus carried TPX2-shR.NA1/2/3/4 and TPX2-NC-shRNA were infected into HeLa cells, respectively. The expression levels of TPX2 mRNA and protein in HeLa cells were determined by Real-time fluorescent quantitative PCR and Western blot, respectively. The recombinant lentivirus was chosen with the best silence effect for the following function experiment. Effect of TPX2-shRNA on cell invasion in HeLa cells was detected using Transwell basementmembrane invasion experimen. Effect of TPX2-shRNA on apoptosis in HeLa cells was detected by flow cytometry （FCM）. The changes of Bcl-2, Bax, Caspase-3, MMP9, TIMP-1 and nm23-H1 protein levels were detected by Western blot. The result showed that optimizing and selecting recombinant lentivirus named LV-TPX2-shRNA-1 could effectively inhibit TPX2 expression in HeLa cells, then LV-TPX2-shRNA-1 was selected for follow- up experiments. In the group of TPX2-shRNA-1, the apoptosis rate was significantly more than those in the control group （P〈0.01）, cells through the basement membrane of transwell chamber were significantly decreased compared to those in the control group （P〈0.01）. Silencing TPX2 expression could up-regulate the levels of Caspase-3, Bax, nm23-H1 and TIMP-1, down-regulate the levels of Bcl-2 and MMP9 in HeLa cells （P〈0.05）. In conclusion, silencing TPX2 expression could increase the apoptosis, which might be related to the up-regulation of the Caspase-3 and Bax, down-regulation of the Bcl-2 in HeLa cells. Silencing TPX2 expression could inhibit the invasion in HeLa cells which might be related to the up-regulation of the nm23-H1 and TIMP-1 levels, down- regulation of the MMP9 levels in HeLa cells.