Wu Chao, Zhao Aili, Tan Tingzhao, Wang Yuan, Shen Zhentao
Department of Medical Oncology, Liaocheng Cancer Prevention and Treatment Hospital, Liaocheng, Shandong 252000, P.R. China.
Radiology Department, Liaocheng People's Hospital, Liaocheng, Shandong 252000, P.R. China.
Exp Ther Med. 2019 Jul;18(1):550-558. doi: 10.3892/etm.2019.7601. Epub 2019 May 23.
Patients with triple negative breast cancer (TNBC) have a poor survival rate following chemotherapy due to drug resistance. Notably, the molecular mechanism of drug resistance remains elusive. Between December 2011 and December 2014, 36 TNBC samples were obtained from Liaocheng People's Hospital. Three gemcitabine-resistant MDA-MB-231 cell lines (MDA-MB-231rGEM1, MDA-MB-231rGEM2 and MDA-MB-231rGEM3) were obtained by exposure of MDA-MB-231 cells to increasing concentrations of gemcitabine for >12 months. Reverse transcription-quantitative polymerase chain reaction was performed to detect the expression levels of specific genes, including microRNA (miR)-620, ATP-binding cassette sub-family B member 1 (ABCB1), ABCC10, cytidine monophosphate kinase, deoxycytidine monophosphate deaminase (DCTD), nucleoside diphosphate kinase 1 (NME1), ribonucleoside-diphosphate reductase large subunit (RRM1) and RRMB2. Western blot analysis was performed to assess the protein expression levels of DCTD. Furthermore, cell proliferation was assessed using a Cell Counting Kit-8 assay and cell apoptosis was detected using an Annexin V/Dead Cell Apoptosis kit. Interactions between miR-620 and DCTD were predicted using TargetScan and detected with the dual luciferase reporter assay. Elevation of miR-620 expression levels were detected in two of the assessed gemcitabine-resistant MDA-MB-231 cell lines compared with MDA-MB-231 cells. Gemcitabine induced significant elevation of miR-620 in MDA-MB-231 cells. An increase of DCTD at mRNA and protein expression levels in MDA-MB-231rGEM1 cells was observed compared with those in MDA-MB-231 cells. Results suggested that DCTD was directly regulated by miR-620. Inhibition of miR-620 and overexpression of DCTD reversed gemcitabine resistance in MDA-MB-231rGEM1 cells via inducing cell apoptosis and cell growth arrest. A negative correlation was identified between miR-620 and DCTD mRNA expression levels in patients with TNBC. The present results demonstrated that overexpression of miR-620 could contribute to the development of gemcitabine resistance in patients with TNBC via the direct downregulation of DCTD.
三阴性乳腺癌(TNBC)患者化疗后的生存率较低,原因是存在耐药性。值得注意的是,耐药的分子机制仍不清楚。在2011年12月至2014年12月期间,从聊城市人民医院获取了36份TNBC样本。通过将MDA-MB-231细胞暴露于浓度不断增加的吉西他滨中超过12个月,获得了三种吉西他滨耐药的MDA-MB-231细胞系(MDA-MB-231rGEM1、MDA-MB-231rGEM2和MDA-MB-231rGEM3)。进行逆转录定量聚合酶链反应以检测特定基因的表达水平,这些基因包括微小RNA(miR)-620、ATP结合盒亚家族B成员1(ABCB1)、ABCC10、胞苷单磷酸激酶、脱氧胞苷单磷酸脱氨酶(DCTD)、核苷二磷酸激酶1(NME1)、核糖核苷二磷酸还原酶大亚基(RRM1)和RRMB2。进行蛋白质印迹分析以评估DCTD的蛋白质表达水平。此外,使用细胞计数试剂盒-8检测法评估细胞增殖,并使用膜联蛋白V/死细胞凋亡试剂盒检测细胞凋亡。使用TargetScan预测miR-620与DCTD之间的相互作用,并通过双荧光素酶报告基因检测法进行检测。与MDA-MB-231细胞相比,在评估的两种吉西他滨耐药的MDA-MB-231细胞系中检测到miR-620表达水平升高。吉西他滨诱导MDA-MB-231细胞中miR-620显著升高。与MDA-MB-231细胞相比,观察到MDA-MB-231rGEM1细胞中DCTD的mRNA和蛋白质表达水平增加。结果表明,DCTD受miR-620直接调控。抑制miR-620和过表达DCTD通过诱导细胞凋亡和细胞生长停滞逆转了MDA-MB-231rGEM1细胞中的吉西他滨耐药性。在TNBC患者中,miR-620与DCTD mRNA表达水平之间存在负相关。目前的结果表明,miR-620的过表达可能通过直接下调DCTD导致TNBC患者吉西他滨耐药性的产生。