lncRNA 178030.2通过TRPS1对三阴性乳腺癌细胞紫杉醇耐药的影响与机制研究

Effect of lncRNA 178030.2 on paclitaxel resistance in triple negative breast cancer cells through TRPS1 and its molecular mechanism

目的:探讨lncRNA 178030.2通过TRPS1对三阴性乳腺癌细胞紫杉醇耐药的影响与机制。方法:采用逐步增加剂量间歇作用的方法诱导三阴性乳腺癌紫杉醇耐药细胞系并命名为MDA-MB-231/R。采用定量PCR和Western blot检测耐药细胞和亲本细胞中lncRNA 178030.2和TRPS1的表达;应用Lipofectamine 2000将lnc RNA 178030.2高表达质粒pcmv-178030.2和对照质粒pcmv转染至MDA-MB-231细胞,分别为高表达组和对照组,定量PCR检测lnc RNA 178030.2水平的变化,分别用定量PCR和Western blot检测TRPS1 m RNA及蛋白水平的变化;RIP实验检测lnc RNA 178030.2是否与TRPS1相结合;MTT法检测MDAMB-231细胞对紫杉醇的敏感性及细胞增殖。应用Lipofectamine 2000将lnc RNA 178030.2小干扰RNA si178030.2和对照si NC转染至MDA-MB-231/R细胞,分别为干扰组和对照组,定量PCR检测lnc RNA178030.2水平的变化,分别用定量PCR和Western blot检测TRPS1 m RNA及蛋白水平的变化;MTT法检测MDA-MB-231/R细胞对紫杉醇的敏感性及细胞增殖。应用Lipofectamine 2000将TRPS1过表达质粒pcmv-TRPS1和对照质粒pcmv转染至MDA-MB-231细胞,分别为高表达组和对照组,分别用定量PCR和Western blot检测两组细胞中TRPS1 m RNA及蛋白水平的表达情况,然后再分别应用Lipofectamine 2000将lnc RNA 178030.2高表达质粒pcmv-178030.2转染入两组细胞,MTT法检测MDA-MB-231细胞对紫杉醇的敏感性及细胞增殖。结果:成功构建在3μg/ml紫杉醇中稳定生长的三阴性乳腺癌耐药细胞系MDAMB-231/R。定量PCR结果显示:lnc RNA 178030.2在紫杉醇耐药细胞系MDA-MB-231/R中的表达明显高于在其亲本细胞MDA-MB-231中的表达;定量PCR和Western blot显示:TRPS1 m RNA和蛋白在紫杉醇耐药细胞系MDA-MB-231/R中的表达明显低于在其亲本细胞系MDA-MB-231中的表达;与对照组相比,高表达lnc RNA 178030.2组的MDA-MB-231细胞中TRPS1表达下降,细胞对紫杉醇的敏感性降低,细胞增殖增强,且lnc RNA 178030.2确实可以与TRPS1相结合;与对照组相比,低表达lnc RNA 178030.2组的MDA-MB-231/R细胞中TRPS1表达升高,细胞对紫杉醇的敏感性升高,细胞增殖减弱;在MDA-MB-231细胞中过表达TRPS1后再过表达lnc RNA 178030.2,其促进紫杉醇耐药、促进细胞增殖的作用也明显减弱。结论:lnc RNA 178030.2促进三阴性乳腺癌细胞MDA-MB-231的紫杉醇耐药,促进细胞增殖,其发生机制可能与下调TRPS1的表达有关。

Objective:To explore the effects and mechanism of lncRNA 178030.2 on paclitaxel resistance in triple negative breast cancer cells through TRPS1.Methods:Paclitaxel-resistant triple negative breast cancer cell line was induced by increasing dose intermittently and named MDA-MB-231/R.Quantitative PCR and Western blot were used to detect the expression of lncRNA 178030.2 and TRPS1 in drug-resistant cells and parental cells.Cells were divided into high expression group and control group,then transfected with pcmv-178030.2 and pcmv with lipofectamine 2000.lncRNA 178030.2 was detected by qRT-PCR,and TRPS1 mRNA and protein levels were detected by qRT-PCR and Western blot,respectively.Whether lncRNA 178030.2 was combined with TRPS1 was detected by RIP.The paclitaxel sensitivity and cell proliferation were tested by MTT.The MDA-MB-231/R cells were divided into low expression group and control group,then transfected with si178030.2 and siNC with lipofectamine 2000.The changes of lncRNA 178030.2 level were detected by quantitative PCR,TRPS1 mRNA and protein levels were detected by quantitative PCR and Western blot,respectively.The paclitaxel sensitivity and cell proliferation were tested by MTT.TRPS1 overexpression and control cell lines were constructed in MMD-MB-231 cells by the transfection of pcmv-TRPS1 and pcmv with lipofectamine 2000.TRPS1 expression was detected by qRT-PCT and Western blot,then pcmv-178030.2 was transfected with lipofectamine 2000 in MDA-MB-231 cells in which TRPS1 was overexpressed,then paclitaxel sensitivity and cell proliferation were tested by MTT.Results:MDA-MB-231/R,a triple negative breast cancer drug-resistant cell line,was successfully constructed in paclitaxel at 3μg/ml.Quantitative PCR results showed that the expression of lncRNA 178030.2 in paclitaxel-resistant cell line MDA-MB-231/R was significantly higher than that in its parent cell MDA-MB-231.Quantitative PCR and Western blot showed that the expression of TRPS1 mRNA and protein in paclitaxel-resistant cell line MDA-MB-231/R was significantly lower than that in its parent cell line MDA-MB-231.Compared with the control group,the expression of TRPS1 in MDA-MB-231 cells in lncRNA 178030.2 high expression group was decreased.Cell sensitivity to paclitaxel decreased,cell proliferation increased,and lncRNA 178030.2 could indeed combine with TRPS1.Compared with the control group,the expression of TRPS1 in MDA-MB-231/R cells in lncRNA 178030.2 low expression group increased,the sensitivity of cells to taxol increased,and the proliferation of cells decreased.The weakened paclitaxel sensitivity and enhanced cell proliferation induced by lncRNA 178030.2 could be reversed by overexpressing TRPS1.Conclusion:lncRNA 178030.2 weakened paclitaxel sensitivity,enhanced cell proliferation in triple negative breast cancer MDA-MB-231 cells,which may act through down-regulation of TRPS1 expression.