摘要
目的探讨转化生长因子-β激活的长链非编码RNA(lncRNA-ATB)促进乳腺癌细胞侵袭转移及对曲妥珠单抗耐药的分子机制。方法选取外科手术切除的15例对曲妥珠单抗耐药的乳腺癌患者的癌组织和癌旁组织,采用荧光定量逆转录聚合酶链反应(RT-PCR)检测癌组织和癌旁组织中lncRNA-ATB的mRNA水平。对患者术后进行为期36个月的随访,采用Kaplan-Meier法绘制lncRNA-ATB的表达与患者无进展生存时间(PFS)和总生存时间(OS)曲线。选取正常乳腺细胞MCF-10A和乳腺癌细胞MCF-7、MDA-MB-435S、T47D及ZR-75-30,采用RT-PCR检测lncRNA-ATB的mRNA水平。小干扰RNA(siRNA)沉默lncRNA-ATB后采用CCK8(细胞计数试剂盒-8)检测细胞对曲妥珠单抗药物敏感性的变化。采用流式细胞术检测曲妥珠单抗诱导的凋亡水平的变化。Transwell和划痕实验检测lncRNA-ATB对乳腺癌细胞侵袭转移的影响。Western blot检测lncRNA-ATB对多药耐药蛋白1(MDR1)以及上皮-间充质转化(EMT)指标(上皮钙黏蛋白和波形蛋白)的影响。结果对曲妥珠单抗耐药的乳腺癌患者的癌组织中lncRNA-ATB的mRNA水平显著高于癌旁组织(P<0.05),且lncRNA-ATB的高表达提示患者生存时间较短(P<0.05);lncRNA-ATB敲除后显著提高细胞对曲妥珠单抗的敏感性,细胞的半数最大抑制浓度(IC 50)显著降低(P<0.05),且曲妥珠单抗诱导的细胞凋亡水平显著升高(P<0.05)。此外,细胞的侵袭和迁移能力显著下降。Western blot结果提示耐药蛋白MDR1的表达水平显著下降(P<0.05),并显著逆转EMT进程。结论lncRNA-ATB在乳腺癌曲妥珠单抗耐药和侵袭转移的发展中发挥重要作用,lncRNA-ATB可能是临床乳腺癌患者预后判断的一个潜在指标和治疗靶点。
Objective To investigate the molecular mechanism of long non-coding RNA(lncRNA-ATB)activated by transforming growth factor to promote invasion and metastasis of breast cancer cells and resistance to trastuzumab.Methods Fifteen patients with breast cancer who were resistant to trastuzumab and underwent surgical resection of the tumor were selected to detect the mRNA levels of lncrNA-ATB in the cancer tissues and adjacent tissues by fluorescence quantitative reverse transcription polymerase chain reaction(RT-PCR).Patients were followed up for 36 months after surgery,and the expression of lncRNA-ATB was plotted with the progression-free survival time(PFS)and total survival time(OS)by Kaplan-Meier method.Normal breast cells MCF-10A and breast cancer cells MCF-7,MDA-MB-435S,T47D and ZR-75-30 were selected to detect the mRNA level of lncRNA-ATB by RT-PCR.After lncRNA-ATB was silenced by small interfering RNA(siRNA),changes in the sensitivity of cells to trastuzumab were detected by CCK8(cell counting kit-8).Flow cytometry was used to detect the apoptosis induced by trastuzumab.The effect of lncRNA-ATB on invasion and metastasis of breast cancer cells was detected by Transwell and wound healing test.Western blot was used to analyze the effects of lncRNA-ATB on multidrug resistant protein 1(MDR1)and epithelial-mesenchymal transformation(EMT)indicators(E-cadherin and vimentin).Results The mRNA level of lncRNA-ATB in cancer tissues of patients with breast cancer was significantly higher than that in the adjacent tissues(P<0.01).The high expression of lncRNA-ATB indicated the shorter survival time of patients(P<0.05).lncRNA-ATB knock out significantly increased the cell sensitivity to trastuzumab,the half maximal inhibitory concentration(IC 50)decreased significantly(P<0.05),and the level of apoptosis induced by trastuzumab was significantly increased(P<0.05).In addition,the ability of cells to invade and migrate was significantly reduced.Western blot results showed that the expression level of drug-resistant protein MDR1 decreased significantly(P<0.05),and the EMT process was significantly reversed.Conclusions lncRNA-ATB plays an important role in the development of trastuzumab resistance and invasive metastasis in breast cancer,and lncRNA-ATB may be a potential prognostic indicator and a potential therapeutic target for clinical breast cancer patients.
作者
庹敏
成宏
周勇
李靖
覃咸雄
于强
彭世军
TUO Min;CHENG Hong;ZHOU Yong;LI Jing;QIN Xianxiong;YU Qiang;PENG Shijun(Department of Breast Surgery,Central Hospital of Enshi Tujia and Miao Autonomous Prefecture,Enshi Tujia and Miao Autonomous Prefecture,Hubei 445000,China)
出处
《徐州医科大学学报》
CAS
2020年第12期870-876,共7页
Journal of Xuzhou Medical University
基金
恩施州科学技术研究与开发项目(D20140012)。
关键词
乳腺癌
长链非编码RNA
曲妥珠单抗
耐药
侵袭转移
breast cancer
long non-coding RNA
trastuzumab
drug resistance
invasion and metastasis
