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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。
The purpose of this study was to investigate the effect and mechanism of lentivirus-mediated targeting protein Xklp2 (TPX2) silencing on apoptosis and invasion of human cervical cancer HeLa cells. Four recombinant lentiviruses carrying TPX2-sh RNA and their negative controls were constructed and five recombinant lentiviruses were stably infected with human cervical cancer HeLa cells. The best TPX2 silencing effect was screened by real-time fluorescence quantitative PCR and Western blot A group of He La cells was used as a follow-up experiment for the interference group. Transwell invasion assay was used to determine the invasiveness of each group of cells. Flow cytometry was used to detect the apoptosis of each group of cells. The levels of Bcl-2, Bax, Caspase-3, MMP9, TIMP-1 and nm23-H1 were detected by Western blot before and after TPX2-sh RNA transfection. The results showed that the RNA interference lentiviral vector LV-TPX2-sh RNA-1 could effectively inhibit the expression of TPX2 in HeLa cells. Compared with the control group, the apoptosis rate of HeLa cells in the interference group was significantly increased (P <0.01 ). The number of cells passing through the basement membrane of Transwell cells was significantly decreased (P <0.01). HeLa cells infected with LV-TPX2-sh RNA-1 could down-regulate the expression of Bcl-2, upregulate the expression of Caspase-3 and Bax (P <0.05), upregulate the expression of invasion related proteins nm23-H1 and TIMP- 1 level, down-regulated the level of MMP9 (P <0.05). The above results show that silencing TPX2 expression can increase the apoptosis of cervical cancer cells, which may be related to its up-regulation of Caspase-3 and Bax levels and down-regulation of Bcl-2 levels; silencing of TPX2 expression can inhibit the invasiveness of cervical cancer cells, And nm23-H1 levels, down-regulated the level of MMP9.