慢病毒介导shRNA沉默乳腺癌MCF-7细胞MTDH基因对紫杉醇敏感性影响的研究

发布时间：2018-09-19 06:43

【摘要】：目的:乳腺癌已成为女性最常见的恶性肿瘤之一。化疗是治疗乳腺癌的主要手段之一,但由于获得性耐药导致化疗失败却是临床治疗中的一个重大挑战。MTDH原癌基因对促进肿瘤细胞的增殖、肿瘤血管生成,引起凋亡抑制有重要作用,并与肿瘤细胞侵袭、扩散、转移及化疗耐药密切相关。本实验通过研究MTDH基因沉默对乳腺癌MCF-7细胞增殖及对紫杉醇耐药性的影响,为乳腺癌的基因治疗及逆转化疗耐药提供实验理论依据。方法:本实验成功构建慢病毒介导MTDH基因沉默的MCF-7稳转细胞株(命名为MCF-7-MTDH/knockdown)和阴性对照病毒稳定转染的乳腺癌细胞株(命名为MCF-7-control)。采用Real-time PCR和Western blot法检测空白对照组(MCF-7)、阴性对照组(MCF-7-control)、实验组(MCF-7-MTDH/knockdown)的MTDH mRNA和蛋白质表达水平验证转染效果。CCK-8法绘制沉默前后MCF-7细胞株的生长曲线,并通过CCK-8法检测沉默MCF-7细胞MTDH基因对紫杉醇药物敏感性的影响。流式细胞术检测沉默前后细胞周期及紫杉醇对细胞周期和凋亡的影响。Real-time PCR和Western blot法检测NF-κB P65,IκBα基因在MCF-7、MCF-7-control、MCF-7-MTDH/knockdown细胞中的mRNA和蛋白质表达水平。通过建立稳定转染细胞的荷瘤裸鼠模型,研究MTDH沉默在体内对紫杉醇敏感性的影响,并对成瘤标本进行流式细胞术检测细胞凋亡情况。应用SPSS21.0统计软件对实验数据进行统计分析,检验以P0.05为差异有显著性意义。每组实验不少于3个独立样本的重复。结果:1慢病毒介导MTDH沉默的乳腺癌MCF-7稳转细胞株的构建和鉴定成功构建稳定转染的MCF-7-MTDH/knockdown细胞株和MCF-7-control细胞株。通过Real-time PCR检测MCF-7细胞、mcf-7-control细胞、mcf-7-mtdh/knockdown细胞中mtdh基因mrna表达水平分别为1.001±0.010、1.014±0.018、0.285±0.036。通过westernblot检测mtdh蛋白在三种细胞中的相对表达量分别为0.871±0.126、0.816±0.067、0.089±0.011。mcf-7-mtdh/knockdown细胞mtdh基因mrna表达和蛋白表达均低于两对照组(p0.05)。2mtdh沉默对细胞增殖和细胞周期、细胞凋亡的影响cck-8法检测各组细胞于0h、24h、48h、72h的增殖情况,实验组mcf-7-mtdh/knockdown细胞增殖速度明显慢于mcf-7和mcf-7-control两对照组(p0.01)。流式细胞仪检测各组的细胞周期,结果显示空白对照组、阴性对照组、实验组细胞g0/g1期比例分别为41.61±0.53%、40.27±1.22%、55.70±1.83%;实验组s期和g2/m期比例均减低。与对照对相比实验组细胞g0/g1期延长,s期和g2/m期缩短(p0.05)。流式细胞学检测各组的细胞凋亡,结果显示:空白对照组、阴性对照组、实验组细胞的凋亡率分别为4.85±0.64%、5.35±0.56%、10.21±0.29%。实验组mcf-7-mtdh/knockdown细胞凋亡率增加,高于对照两组(p0.05)。3紫杉醇对mtdh基因沉默前后mcf-7细胞增殖和周期、细胞凋亡的影响0.1μg/ml紫杉醇作用48h对三组细胞的抑制率分别为40.71±0.08%、40.96±0.16%、54.25±0.06%;1μg/ml紫杉醇作用48h对三组细胞的抑制率分别为56.54±0.13%、56.49±0.04%、77.19±0.17%。0.1μg/ml、1μg/ml紫杉醇作用48h后对实验组mcf-7-mtdh/knockdown细胞的抑制率均高于两对照组(p0.01)。细胞周期检测结果显示:紫杉醇药物可诱导各组细胞g2/m期比例升高,1μg/ml用药组较0.1μg/ml用药组g2/m期比例升高更加明显(p0.05);1μg/ml紫杉醇作用48h后mcf-7-mtdh/knockdown细胞的g2/m期比例明显高于mcf-7、mcf-7-control细胞(p0.05)。细胞凋亡情况检测结果显示:各组细胞凋亡率随紫杉醇浓度增加而增加。0.1μg/ml和1μg/ml紫杉醇分别作用48h后,实验组mcf-7-mtdh/knockdown细胞的凋亡率明显高于两对照组(p0.001)。4沉默前后MCF-7细胞NF-κB P65,IκBα基因的表达情况Real-time PCR和Western blot结果显示:MTDH沉默后P65的mRNA和蛋白表达量降低,而IκBα的mRNA和蛋白表达量升高(P0.001)。与对照组相比差异有统计学意义。5 MTDH沉默对裸鼠成瘤的影响种植瘤的生长情况:MCF-7-MTDH/knockdown组裸鼠的肿瘤体积(374.35±16.68mm3 vs 902.7±26.53mm3 and 840.79±25.82mm3)和重量(0.459±0.051g vs 1.106±0.095g and 1.103±0.101g)明显小于两对照组(P0.001),生长速度较对照组明显减慢。种植瘤对紫杉醇敏感性:MCF-7-MTDH/knockdown组裸鼠肿瘤体积(91.13±16.68 mm3 vs 340.21±26.35mm3 and 322.27±25.82 mm3)和重量(0.113±0.02 vs 0.440±0.01 g and 0.397±0.03 g)明显小于两对照组(P0.001)。流式细胞术检测种植瘤的细胞凋亡:未应用紫杉醇的MCF-7、MCF-7-control、MCF-7-MTDH/knockdown三组种植瘤细胞凋亡率分别为2.477±0.055%、2.823±0.770%、9.527±0.262%。MCF-7-MTDH/knockdown组裸鼠种植瘤凋亡率增加(P0.001)。应用紫杉醇后三组种植瘤细胞凋亡率分别为14.547±0.625%、13.320±1.529%、25.613±0.996%。MCF-7-MTDH/knockdown组裸鼠种植瘤凋亡率较两实验组明显增加(P0.001)。结论:1通过慢病毒介导能够成功构建MTDH沉默的MCF-7稳定转染细胞株。2 MTDH沉默可抑制乳腺癌MCF-7细胞增殖,诱导乳腺癌MCF-7细胞G0/G1期比例升高,促进细胞凋亡。3 MTDH沉默可提高乳腺癌细胞对紫杉醇的敏感性,其分子机制可能与NF-κB/IκB通路抑制有关。
[Abstract]:Objective: Breast cancer has become one of the most common malignant tumors in women. Chemotherapy is one of the main methods for the treatment of breast cancer, but failure of chemotherapy due to acquired drug resistance is a major challenge in clinical treatment. Invasion, diffusion, metastasis and chemotherapeutic resistance of breast cancer cells are closely related. This study was designed to investigate the effect of MTDH gene silencing on the proliferation and paclitaxel resistance of breast cancer MCF-7 cells, and to provide experimental theoretical basis for gene therapy of breast cancer and reversal of chemotherapeutic resistance. The expression levels of MTDH mRNA and protein in MCF-7-MTDH/knockdown and MCF-7-control were detected by Real-time PCR and Western blot. CCK-8 method was used to plot the growth curve of MCF-7 cell line before and after silencing, and CCK-8 method was used to detect the effect of MTDH gene on the drug sensitivity of paclitaxel. Flow cytometry was used to detect the cell cycle before and after silencing and the effect of paclitaxel on cell cycle and apoptosis. Expression levels of mRNA and protein in MCF-7, MCF-7-control and MCF-7-MTDH/knockdown cells were measured by flow cytometry. The effect of MTDH silencing on paclitaxel sensitivity in vivo was studied by establishing a stable tumor-bearing nude mice model. Results: 1. Lentivirus-mediated MTDH silencing breast cancer MCF-7 stable transfection cell line was successfully constructed and identified. The stable transfected MCF-7-MTDH/knockdown cell line and MCF-7-control cell line were successfully constructed by Real-time PCR. The expression levels of mtdh gene mRNA in MCF-7 cells, mcf-7-mtdh/knockdown cells and mcf-7-mtdh/knockdown cells were 1.001 (+ 0.010), 1.014 (+ 0.018) and 0.285 (+ 0.036) respectively. The relative expression levels of mtdh protein in the three kinds of cells were 0.871 (+ 0.126), 0.816 (+ 0.067), 0.089 (+ 0.011) mtdh/knockdown cells by Western blot, respectively. The expression and protein expression of mcf-7-mtdh / knockdown cells were significantly slower than those of MCF-7 and mcf-7-control cells (p0.01). Cell cycle, the results showed that the blank control group, the negative control group, the experimental group G0 / G1 phase ratio was 41.61 + 0.53%, 40.27 + 1.22%, 55.70 + 1.83%; experimental group S phase and G2 / M phase ratio were reduced. compared with the control group, the experimental group cells G0 / G1 phase prolonged, S phase and G2 / M phase shortened (p0.05). flow cytometry detection of cell apoptosis, node The results showed that the apoptosis rate of mcf-7-mtdh/knockdown cells in the experimental group was higher than that in the control group (p0.05). The inhibitory rates of paclitaxel at 48 h were 40.71 (+ 0.08%), 40.96 (+ 0.16%), 54.25 (+ 0.06%) and 56.54 (+ 0.13%), 56.49 (+ 0.04%) and 77.19 (+ 0.17%) respectively. The results of cell cycle test showed that the proportion of G2 / M phase of mcf-7-mtdh / knockdown cells was significantly higher than that of MCF-7 and mcf-7-control cells (p0.05). The percentage of G2 / M phase of mcf-7-mtdh / knockdown cells was significantly higher in 1 ug / ml group than that in 0.1 UG / ml group (p0.05). The results showed that the apoptosis rate of MCF-7 cells increased with the increase of paclitaxel concentration. After 48 hours of treatment with 0.1 ug/ml and 1 ug/ml paclitaxel respectively, the apoptosis rate of mcf-7-mtdh/knockdown cells in the experimental group was significantly higher than that in the control group (p0.001). 4 The expression of NF-kappa B P65 and I-kappa B alpha gene in MCF-7 cells before and after silencing was significantly higher than that in the control group (p0.001). After TDH silencing, the expression of P65 mRNA and protein decreased, while the expression of I-kappa Balpha mRNA and protein increased (P 0.001). The difference was statistically significant compared with the control group. 5 MTDH silencing on the growth of implanted tumors in nude mice: MCF-7-MTDH/knockdown group nude mice tumor volume (374.35+16.68mm3 vs 902.7+26.53mm3 and 840.79+25.82m) M3 and weight (0.459 + 0.051g vs 1.106 + 0.095g and 1.103 + 0.101g) were significantly lower than those of the two control groups (P 0.001), and the growth rate was significantly slower than that of the control group. Flow cytometry was used to detect the apoptosis of implant tumors: the apoptosis rates of MCF-7, MCF-7-control, MCF-7-MTDH/knockdown groups were 2.477 [0.055], 2.823 [0.770], 9.527 [0.262]. The apoptosis rates of implant tumors in MCF-7-MTDH/knockdown group were 2.477 [0.055], 2.823 [0.770], 9.527 [0.262], respectively. The apoptotic rates of the three groups were 14.547 (+ 0.625%), 13.320 (+ 1.529%) and 25.613 (+ 0.996%) respectively. The apoptotic rates of the nude mice in MCF-7-MTDH / knockdown group were significantly higher than those in the two experimental groups (P 0.001). Conclusion: 1. MTDH silencing MCF-7 stable transfected cell lines could be successfully constructed by lentivirus mediation. Proliferation of breast cancer MCF-7 cells, induction of G0/G1 phase ratio of breast cancer MCF-7 cells, and promotion of apoptosis. 3 MTDH silencing can enhance the sensitivity of breast cancer cells to paclitaxel, and its molecular mechanism may be related to the inhibition of NF-kappa B/I kappa B pathway.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.9

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