Lin Sen, Wang Hongbo, Yang Wenjuan, Wang Aiguang, Geng Chao
Department of Gastroenterology, The Second Hospital of Shandong University, Jinan City, Shangdong 250033, People's Republic of China.
Department of Nursing, Jinan Central Hospital, Jinan City, Shangdong 250013, People's Republic of China.
Onco Targets Ther. 2020 Jan 13;13:337-349. doi: 10.2147/OTT.S220302. eCollection 2020.
This study aimed to evaluate the specific role of colon cancer-associated transcript 2 (CCAT2) on gastric cancer (GC), and reveal the potential regulatory mechanism relating to mammalian target of rapamycin (mTOR) signaling.
The expression of CCAT2 was detected in GC tissues and cells by quantitative real-time PCR (qRT-PCR), and its relation with the pathologic characteristics of GC patients was analyzed. HGC-27 and SGC-7901 cells were transfected with siRNA-CCAT2 to silence CCAT2, and HGC-27 cells were then treated with an mTOR agonist Leucine (Leu) to activate mTOR signaling. The cell proliferation was evaluated by cell viability and colony formation. The cell cycle and apoptosis, and the migration and invasion abilities were detected by Flow cytometry, and Transwell assay, respectively. The expression of PCNA (proliferation marker), Snail, N-cadherin, E-cadherin (invasion markers), P53, Caspase-8, Bcl-2 (apoptosis markers), LC3-II/LC3-I, ATG3, p62 (autophagy makers), phosphorylated mTOR (p-mTOR), p-AKT, and p-p70S6K (mTOR signaling markers) were detected by Western blot.
CCAT2 was upregulated in GC tissues and cells, and positively associated with the maximum tumor diameter, lymphatic metastasis, TNM staging, and low overall survival rate (P < 0.05). siRNA-CCAT2 transfection significantly inhibited the viability, colony formation, and migration and invasion abilities, blocked the cell cycle in G0/G1 phase, and promoted the apoptosis and autophagy of SGC-7901 and HGC-27 cells (P < 0.05). In addition, siRNA-CCAT2 transfection significantly upregulated P53, Caspase-8, LC3-II/LC3-I and ATG3, and downregulated PCNA, Bcl-2, p62, p-mTOR, p-AKT and p-p70S6K in SGC-7901 and HGC-27 cells (P < 0.05). siRNA-CCAT2 reversed the tumor-promoting effect of mTOR signaling activation on HGC-27 cells (P < 0.05).
Silencing of CCAT2 inhibited the proliferation, migration and invasion, and promoted the apoptosis and autophagy of GC cells through blocking mTOR signaling.
本研究旨在评估结肠癌相关转录本2（CCAT2）在胃癌（GC）中的具体作用，并揭示与雷帕霉素靶蛋白（mTOR）信号传导相关的潜在调控机制。
采用定量实时聚合酶链反应（qRT-PCR）检测GC组织和细胞中CCAT2的表达，并分析其与GC患者病理特征的关系。用小干扰RNA（siRNA）-CCAT2转染HGC-27和SGC-7901细胞以沉默CCAT2，然后用mTOR激动剂亮氨酸（Leu）处理HGC-27细胞以激活mTOR信号传导。通过细胞活力和集落形成评估细胞增殖。分别用流式细胞术和Transwell实验检测细胞周期、凋亡以及迁移和侵袭能力。通过蛋白质免疫印迹法检测增殖标志物增殖细胞核抗原（PCNA）、侵袭标志物Snail、N-钙黏蛋白、E-钙黏蛋白、凋亡标志物P53、半胱天冬酶-8、Bcl-2、自噬标志物微管相关蛋白1轻链3-II/微管相关蛋白1轻链3-I（LC3-II/LC3-I）、自噬相关蛋白3（ATG3）、p62、磷酸化mTOR（p-mTOR）、磷酸化蛋白激酶B（p-AKT）和磷酸化核糖体蛋白S6激酶（p-p70S6K）（mTOR信号传导标志物）的表达。
CCAT2在GC组织和细胞中上调，且与最大肿瘤直径、淋巴转移、TNM分期及低总生存率呈正相关（P<０.０５）。siRNA-CCAT2转染显著抑制SGC-7901和HGC-27细胞的活力、集落形成以及迁移和侵袭能力，使细胞周期阻滞在G0/G1期，并促进细胞凋亡和自噬（P<０.０５）。此外，siRNA-CCAT2转染显著上调SGC-7901和HGC-27细胞中P53、半胱天冬酶-8、LC3-II/LC3-I和ATG3的表达，下调PCNA、Bcl-2、p62、p-mTOR、p-AKT和p-p70S6K的表达（P<０.０５）。siRNA-CCAT2可逆转mTOR信号传导激活对HGC-27细胞的促肿瘤作用（P<０.０５）。
沉默CCAT2可通过阻断mTOR信号传导抑制GC细胞的增殖、迁移和侵袭，并促进其凋亡和自噬。
