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目的:探讨下调中介体(mediator,Med)19表达是否增加紫杉醇(pacliatxel,PTX)对p53野生型和突变型乳腺癌细胞化疗的疗效。方法:构建Med19 RNA干扰(RNAi)慢病毒,分别感染p53野生型的乳腺癌细胞MCF-7和p53突变型的乳腺癌细胞MDA-MB-231,两种细胞均分为Med19基因敲减组(KD组:细胞感染pGcscil-Med19-si RNA-GFP)、空载体感染组(NC组:细胞感染pGcscil-Med19-NC-GFP)、对照组(CON组:未感染慢病毒的乳腺癌细胞),荧光显微镜观察慢病毒感染效率。Western blotting检测Med19基因沉默后各组细胞内P53、磷酸化P53(p P53)、P21蛋白表达水平的变化。紫杉醇分别作用于Med19基因沉默前后的MCF-7和MDA-MB-231细胞,MTT法检测细胞增殖抑制率的变化,流式细胞术检测细胞周期和凋亡率的变化。结果:KD组和NC组细胞的荧光感染效率均大于90%,表明获得了满意的感染效率。与CON组和NC组相比,两种细胞KD组的Med19蛋白表达水平均下降,差异有统计学意义(均P<0.05)。MCF-7细胞KD组P53、p P53、P21蛋白表达上调,差异有统计意义(均P<0.05)。在0.01~50μg/ml范围内,紫杉醇抑制两种乳腺癌细胞的生长增殖并呈浓度依赖性(P<0.05),对KD组的抑制率和细胞凋亡率均高于NC组及CON组,差异有统计学意义(均P<0.05);但MDA-MB-231细胞各组间抑制率差异无统计学意义(均P>0.05)。10μg/ml紫杉醇导致两种细胞的KD组和NC组均出现G_2/M期阻滞,并且MCF-7细胞KD组G_0/G_1期细胞较NC组显著增加(P<0.05)。结论:Med19基因沉默增强紫杉醇对p53野生型乳腺癌MCF-7细胞化疗的疗效,其机制可能为增强了p53基因的表达活化,通过其下游基因p21调节细胞周期、促进细胞凋亡。
OBJECTIVE: To investigate whether down-regulation of mediator 19 can increase the efficacy of paclitaxel (PTX) in the chemotherapy of p53 wild-type and mutant breast cancer cells. Methods: Med19 RNA interference lentivirus was transfected into MCF-7 cells of p53 wild type and MDA-MB-231 cells of p53 mutant type. The two cell lines were divided into Med19 knockdown group (NC group: cell-infected pGcscil-Med19-NC-GFP), control group (CON group: lentivirus-free breast cancer cells), control group Observe the lentiviral infection efficiency by fluorescence microscopy. Western blotting was used to detect the expression of P53, phosphorylated P53 (P P53) and P21 in each group after Med19 gene silencing. Paclitaxel, respectively, before and after Med19 gene silencing MCF-7 and MDA-MB-231 cells, MTT assay of cell proliferation inhibition rate changes, flow cytometry to detect changes in cell cycle and apoptosis rate. Results: Fluorescent infection efficiencies of KD group and NC group were more than 90%, indicating that satisfactory infection efficiency was obtained. Compared with CON group and NC group, the expression levels of Med19 in both KD groups decreased significantly (all P <0.05). The expression of P53, p P53, P21 in MCF-7 KD group was up-regulated, the difference was statistically significant (P <0.05). In the range of 0.01 ~ 50μg / ml, paclitaxel inhibited the growth and proliferation of both breast cancer cells in a concentration-dependent manner (P <0.05), and the inhibitory rate and apoptosis rate in KD group were higher than that in NC group and CON group The difference was statistically significant (all P <0.05). However, the inhibitory rates of MDA-MB-231 cells in each group had no significant difference (all P> 0.05). Paclitaxel at 10μg / ml resulted in G 2 / M arrest in both KD and NC groups, and the number of G 0 / G 1 cells in KD group increased significantly compared with NC group (P <0.05). Conclusion: Med19 gene silencing enhances the efficacy of paclitaxel in the chemotherapy of p53 wild-type breast cancer MCF-7 cells. Its mechanism may be to enhance the expression and activation of p53 gene and regulate its cell cycle through its downstream gene p21.