摘要
目的:探讨磷脂酰肌醇特异性磷脂酶Cε(phosphoinositide-speci c phospholipase Cε,PLCε)对前列腺癌细胞增殖和比卡鲁胺敏感性的影响及其对Wnt/β-catenin信号通路的调控作用。方法:采用比卡鲁胺浓度递增及间歇诱导法建立对比卡鲁胺耐药的前列腺癌B-LNCAP细胞。应用实时荧光定量PCR和蛋白质印迹法检测前列腺癌LNCAP和B-LNCAP细胞中雄激素受体(androgen receptor,AR)和PLCεmRNA和蛋白的表达,CCK-8法检测LNCAP和B-LNCAP细胞对比卡鲁胺的敏感性。将干扰PLCε表达的重组慢病毒LV-shPLCε或阴性对照(negative control,NC)LV-NC感染B-LNCAP细胞(称为LVshPLCε/B-LNCAP或LV-NC/B-LNCAP),Wnt/β-catenin信号通路激活剂AZD2858处理LV-shPLCε/B-LNCAP细胞,以未进行任何干预的B-LNCAP细胞作为空白组。应用CCK-8法检测各组细胞的增殖情况及对比卡鲁胺的敏感性,应用克隆形成实验检测各组细胞的克隆形成能力,蛋白质印迹法检测各组细胞的细胞质和细胞核中β-catenin和AR蛋白表达水平,实时荧光定量PCR和蛋白质印迹法检测各组细胞中c-myc、cyclin D1mRNA和蛋白表达水平。结果:B-LNCAP细胞中PLCε、AR mRNA和蛋白的表达水平均高于LNCAP细胞(P值均<0.05),B-LNCAP细胞对比卡鲁胺的耐药系数为132.87,成功构建前列腺癌耐药细胞B-LNCAP。LV-shPLCε/B-LNCAP细胞增殖和克隆形成能力均弱于空白组和LV-NC/B-LNCAP组(P值均<0.01),比卡鲁胺对LV-shPLCε/B-LNCAP细胞的半数抑制浓度(half maximal inhibitory concentration,IC50)值均低于空白组和LV-NC/B-LNCAP组(P值均<0.05),LV-shPLCε/B-LNCAP细胞核中β-catenin和AR蛋白表达水平明显低于空白组和LV-NC/B-LNCAP组(P值均<0.01),LVshPLCε/B-LNCAP细胞中c-myc、cyclin D1 mRNA和蛋白的表达水平均低于空白组和LV-NC/B-LNCAP组(P值均<0.01);AZD2858处理的LVshPLCε/B-LNCAP细胞增殖和克隆形成能力强于LV-shPLCε/B-LNCAP细胞(P值均<0.05),比卡鲁胺对AZD2858处理的LV-shPLCε/B-LNCAP细胞IC50值高于未处理的LV-shPLCε/B-LNCAP细胞(P<0.05),AZD2858处理的LV-shPLCε/B-LNCAP细胞核中β-catenin和AR蛋白表达水平均高于LV-shPLCε/B-LNCAP细胞(P值均<0.05),AZD2858处理的LV-shPLCε/B-LNCAP细胞中c-myc、cyclin D1 mRNA和蛋白表达水平均高于LV-shPLCε/B-LNCAP细胞(P值均<0.05)。结论:PLCε下调后通过抑制Wnt/β-catenin信号通路,增强前列腺癌B-LNCAP细胞对比卡鲁胺的敏感性,抑制前列腺癌B-LNCAP细胞的增殖。
Objective： To investigate the effects of phosphoinositide-specific phospholipase Cε（PLCε） on the proliferation and sensitive of prostate cancer cells to bicalutamide as well as the regulation of Wnt/β-catenin signaling pathway.Methods： Bicalutamide-resistant prostate cancer B-LNCAP cells were constructed by increasing concentration of bicalutamide in a stepwise and intermittent manner. The expressions of androgen receptor（AR） and PLCε mRNA and protein in LNCAP and B-LNCAP cells were detected by real-time fluorescent quantitative PCR and Western blotting, respectively. The sensitivity of LNCAP and B-LNCAP cells to bicalutamide was detected by CCK-8 assay. The recombination lentivirus interfering PLCε expression（LV-shPLCε） and its negative control lentivirus（LV-NC） were used to infect B-LNCAP cells（named as LV-shPLCε/B-LNCAP or LV-NC/B-LNCAP）, respectively.LV-shPLCε/B-LNCAP cells were treated with Wnt/β-catenin signaling pathway activator AZD2858,while B-LNCAP cells without any intervention was used as a blank group. Then the sensitivity of cells to bicalutamide and the cell proliferation in each group were tested by CCK-8 assay.The cell clonality in each group was tested by clonal formation assay. The expressions ofβ-catenin and AR proteins in cell cytoplasm and nucleus were detected by Western blotting. The expressions of c-myc and cyclin D1 mRNA and protein in each group were detected by real-time fluorescent quantitative PCR and Western blotting, respectively.Results： The expression levels PLCε and AR mRNA and protein in B-LNCAP cells were statistically higher than those in LNCAP cells（all P〈0.05）. The drug resistance index of B-LNCAP cells to bicalutamide was 132.87, which indicated that the drug-resistant prostate cancer B-LNCAP cells were successfully constructed. The clone formation and cell proliferation abilities of LV-shPLCε/B-LNCAP cells were lower than those of the blank and LV-NC/B-LNCAP groups（all P〈0.01）. The half maximal inhibitory concentration（IC50） value of bicalutamide in LV-shPLCε/B-LNCAP cells was lower than that of the blank or LV-NC/B-LNCAP groups（both P〈0.05）. The expression levels of nuclear protein β-catenin and AR in LV-shPLCε/B-LNCAP cells were decreased as compared with the blank and LV-NC/B-LNCAP groups（all P〈0.01）. The expression levels of c-myc and cyclin D1 mRNA and protein in LV-shPLCε/B-LNCAP cells were decreased as compared with the blank and LV-NC/B-LNCAP groups（all P〈0.01）. The abilities of clone formation and proliferation of LV-shPLCε/B-LNCAP cells treated with AZD2858 were stronger than those of untreated LV-shPLCε/B-LNCAP cells（both P〈0.05）. The IC50 value of AZD2858 in LV-shPLCε/B-LNCAP cells was increased as compared with untreated LV-shPLCε/B-LNCAP cells（P〈0.05）. The expression levels of nuclear protein β-catenin and AR, as well as the expression levels of c-myc and cyclin D1 mRNA and protein in LV-shPLCε/B-LNCAP cells treated with AZD2858 were higher than those in untreated LV-shPLCε/B-LNCAP cells（all P〈0.05）.Conclusion： Down-regulation of PLCε enhances the sensitivity of prostate cancer B-LNCAP cells to bicalutamide, and inhibits the cell proliferation through suppressing Wnt/β-catenin signaling pathway.
作者
李罗
范佳鑫
牛凌芳
范砚茹
高英英
张尧
罗春丽
LI Luo;FANJiaxin;NIU Lingfang;FAN Yanru;GAO Yingying;ZHANG Yao;LUO Chunli(Key Laboratory of Clinical Diagnostics Founded by Ministry of Education,College of Laboratory,Chongqing Medical University,Chongqing 400016,China;Department of Urinary Surgery,First Affiliated Hospital of Chongqing Medical University,Chongqing 400016.China)
出处
《肿瘤》
CAS
CSCD
北大核心
2018年第8期761-771,791,共12页
Tumor
