Prrx1诱导上皮-间质转化促进乳腺癌细胞对多西他赛耐药的研究
发布时间：2019-06-17 12:45
【摘要】：【目的】探讨同源异型核基因1(Prrx1)诱导上皮-间质转化(EMT)促进乳腺癌细胞对多西他赛(Docetaxel)耐药的影响。【方法】1.用Real-time PCR方法从MCF-7、BT-549和MDA-MB-468三种乳腺癌细胞中筛选出Prrx1低表达乳腺癌细胞株。2.用定制的慢病毒载体感染筛选出的Prrx1低表达乳腺癌细胞株,Real-time PCR验证感染成功后,分别于荧光显微镜的白光和荧光下观察各组细胞形态变化。运用Real-time PCR检测波形蛋白与E-钙黏蛋白的表达。3.用Transwell法检测Prrx1过表达对MCF-7细胞迁移能力的影响。4.CCK-8检测MCF-7细胞的IC50,并检测Docetaxel作用于实验组、空白对照组和阴性对照组24h后的IC50。5运用流式细胞术检测Docetaxel作用于三组细胞24h后,Prrx1过表达对乳腺癌MCF-7细胞凋亡率的影响。6.Western blot检测三组细胞多药耐药相关蛋白1(ABCB1/p-gp)和Twist1的表达。【结果】三种细胞中,Prrx1相对表达量最低的是MCF-7细胞(p(27)0.001)。用Prrx1过表达慢病毒感染MCF-7细胞后,细胞形态由“铺路石样”变成长梭形,并且荧光效率高达80%以上。Real-time PCR结果显示Prrx1m RNA相对表达量是转染前252.09倍(t=44.30,p(27)0.001),波形蛋白是转染前2.65倍(t=12.05,p(27)0.001),E-钙黏蛋白是转染前0.64倍(t=4.91,p(27)0.001)。Transwell结果显示Prrx1过表达的MCF-7细胞发生迁移的细胞数(71.69±6.66)明显高于阴性对照组(40.87±4.16)(t=15.05,p(27)0.001)和空白对照组(41.2±4.62)(15.76,p(27)0.001),差异有统计学意义,而两对照组之间比较差异无统计学意义(t=0.21,p=0.84)。CCK-8结果显示Docetaxel作用MCF-7细胞24h和48h后的IC50值分别是(10.94±0.64)、(10.66±1.68),差异无统计学意义(t=0.27,p=0.80)。慢病毒感染后,Prrx1过表达的MCF-7细胞的IC50明显高于空白对照组(13.30,p(27)0.001)和阴性对照组(t=0.91,p(27)0.001),且差异有统计学意义,而两对照组之间比较差异无统计学意义(t=0.22,p=0.83)。流式细胞术结果显示Docetaxel处理三组细胞24h后Prrx1过表达的MCF-7细胞的凋亡率明显低于阴性对照组(t=9.32,p(27)0.001)和空白对照组(t=9.70,p(27)0.001),差异有统计学意义,而两对照组之间比较差异无统计学意义(t=0.48,p=0.65)。Western blot结果显示:Prrx1过表达MCF-7细胞的Twist1和多药耐药相关蛋白1(MDR1,ABCB1,P-gp)相对表达量明显高于两对照组,且差异有统计学意义(p(27)0.001),而两对照组之间比较差异无统计学意义(p(29)0.05)。【结论】Prrx1诱导EMT促进乳腺癌细胞对Docetaxel耐药。
[Abstract]:[objective] to investigate the effect of homologous nuclear gene 1 (Prrx1) induced epithelial-stroma transformation (EMT) on drug resistance of breast cancer cells to docetaxel (Docetaxel). [methods] 1. Prrx1 low expression breast cancer cell lines were screened out by Real-time PCR from three kinds of breast cancer cells, MCF-7,BT-549 and MDA-MB-468. Prrx1 low expression breast cancer cell lines were screened by custom lentivirus vector infection. After the infection was verified by Real-time PCR, the morphological changes of each group were observed under the white light and fluorescence of fluorescence microscope, respectively. The expression of vimentin and E-cadherin was detected by Real-time PCR. The effect of Prrx1 overexpression on the migration ability of MCF-7 cells was detected by Transwell assay. 4. IC50, of MCF-7 cells was detected by CCK-8 and Docetaxel was detected in the experimental group. IC50.5 in the blank control group and negative control group was detected by flow cytometry after 24 hours of Docetaxel treatment. The effect of Prrx1 overexpression on apoptosis rate of breast cancer MCF-7 cells. 6. Western blot detected the expression of multi-drug resistance associated protein 1 (ABCB1/p-gp) and Twist1 in three groups of cells. [results] among the three groups, the relative expression of Prrx1 was the lowest in MCF-7 cell line (p (27) 0.001). After MCF-7 cells were infected with lentivirus with Prrx1, the morphology of MCF-7 cells changed from "paving stone" to long fusiform, and the fluorescence efficiency was more than 80%. Real-time PCR results showed that the relative expression of Prrx1m RNA was 252.09 times (t 鈮，
本文编号：2500997
[Abstract]:[objective] to investigate the effect of homologous nuclear gene 1 (Prrx1) induced epithelial-stroma transformation (EMT) on drug resistance of breast cancer cells to docetaxel (Docetaxel). [methods] 1. Prrx1 low expression breast cancer cell lines were screened out by Real-time PCR from three kinds of breast cancer cells, MCF-7,BT-549 and MDA-MB-468. Prrx1 low expression breast cancer cell lines were screened by custom lentivirus vector infection. After the infection was verified by Real-time PCR, the morphological changes of each group were observed under the white light and fluorescence of fluorescence microscope, respectively. The expression of vimentin and E-cadherin was detected by Real-time PCR. The effect of Prrx1 overexpression on the migration ability of MCF-7 cells was detected by Transwell assay. 4. IC50, of MCF-7 cells was detected by CCK-8 and Docetaxel was detected in the experimental group. IC50.5 in the blank control group and negative control group was detected by flow cytometry after 24 hours of Docetaxel treatment. The effect of Prrx1 overexpression on apoptosis rate of breast cancer MCF-7 cells. 6. Western blot detected the expression of multi-drug resistance associated protein 1 (ABCB1/p-gp) and Twist1 in three groups of cells. [results] among the three groups, the relative expression of Prrx1 was the lowest in MCF-7 cell line (p (27) 0.001). After MCF-7 cells were infected with lentivirus with Prrx1, the morphology of MCF-7 cells changed from "paving stone" to long fusiform, and the fluorescence efficiency was more than 80%. Real-time PCR results showed that the relative expression of Prrx1m RNA was 252.09 times (t 鈮，
本文编号：2500997
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