Role of -glutamyl cyclotransferase as a therapeutic target for colorectal cancer based on the lentivirus-mediated system
Role of -glutamyl cyclotransferase as a therapeutic target for colorectal cancer based on the lentivirus-mediated system
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DOI：
10.1097/cad.0000000000000407
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发表时间：
2016-11-01
影响因子：
2.3
通讯作者：
Li, Yue
中科院分区：
文献类型：
--
作者：
Dong, Jian;Zhou, Yuanhang;Li, Yue
人GGCT（-谷氨酰环转移酶）已被证明是上调，在大多数肿瘤，但其在结直肠癌（CRC）的作用知之甚少。因此，选择包括HCT 116和SW 1116在内的CRC细胞系，通过使用慢病毒介导的RNA干扰构建GGCT沉默细胞模型来研究GGCT的作用。通过逆转录-定量PCR和蛋白质印迹分析证实敲低效率。通过MTT法、集落形成实验和流式细胞仪分析，研究GGCT沉默对大肠癌细胞的生物学功能。此外，蛋白质印迹和Pathscan细胞内信号传导用于检测与GGCT敲低诱导的细胞生长和凋亡相关的细胞内信号传导。此外，使用临床化疗药物5-氟尿嘧啶通过膜联蛋白V/7-AAD双染色测定来研究GGCT沉默对药物敏感性的影响。分析结果表明，GGCT沉默通过调节p21、p27和cyclin E的表达，显著抑制细胞增殖，并将细胞周期阻滞在G 0/G1期。此外，GGCT沉默通过激活caspase-3和裂解的聚ADP-核糖聚合酶途径以及下调磷酸化富含脯氨酸的Akt底物40 kDa（PRAS 40）表达水平来触发CRC细胞凋亡。此外，GGCT沉默联合5-氟尿嘧啶处理进一步诱导CRC细胞凋亡率。这些结果表明，GGCT可能是一个有前途的诊断和治疗的CRC通过激活凋亡途径的目标。
Human GGCT (-glutamyl cyclotransferase) has been shown to be upregulated in most tumors, but its role in colorectal cancer (CRC) is poorly understood. Thus, CRC cell lines, including HCT116 and SW1116, were chosen to investigate the role of GGCT by constructing a GGCT silencing cells model using lentivirus-mediated RNA interference. The knockdown efficiency was confirmed by reverse transcription-quantitative PCR and a western blot assay. Then, a set of biological functions of GGCT silencing on CRC cell was assessed by MTT, colony-formation assay, and flow cytometry analysis. Further, western blot and Pathscan intracellular signaling were used to detect intracellular signaling associated with cell growth and apoptosis induced by GGCT knockdown. In addition, the clinical chemotherapeutic drug 5-fluorouracil was used to investigate the impact of GGCT silencing on drug sensitivity by an Annexin V/7-AAD double-staining assay. The results of the analysis indicated that GGCT silencing significantly suppressed cell proliferation and arrested cell cycle at the G0/G1 phase by regulating the expression of p21, p27, and cyclin E. Moreover, GGCT silencing triggered the apoptosis of CRC cells by activating caspase-3 and cleaved poly-ADP-ribose polymerase pathways and downregulating the phosphorylation proline-rich Akt substrate of 40kDa (PRAS40) expression levels. Furthermore, GGCT silencing combined with 5-fluorouracil treatment further induced the apoptotic rate of CRC cells. These findings suggest that GGCT may be a promising diagnostic and therapeutic target for CRC by activating the apoptotic pathway.