Zhang Mei-Ying, Wang Li-Yuan, Zhao Shuang, Guo Xiao-Chong, Xu Ying-Qi, Zheng Zhi-Hong, Lu Hang, Zheng Hua-Chuan
Laboratory Animal Center, China Medical University, Shenyang, Liaoning 110001, P.R. China.
Department of Biochemistry and Molecular Biology, College of Basic Medicine, China Medical University, Shenyang, Liaoning 110001, P.R. China.
Oncol Lett. 2019 Feb;17(2):2441-2450. doi: 10.3892/ol.2018.9817. Epub 2018 Dec 10.
Beclin 1 is involved in autophagy, differentiation, apoptosis and cancer progression, and functions as a haploinsufficient tumor suppressor gene. The aim of the present study was to elucidate the function of Beclin 1 in colon cancer. A Beclin 1-expressing plasmid was transfected into HCT-15 and HCT-116 cells, and the phenotypes and associated molecules were determined. Beclin 1 transfectants were subcutaneously injected into nude mice to determine tumor growth, and proliferation and apoptosis levels using Ki-67 immunostaining and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL), respectively. Beclin 1 overexpression inhibited viability as determined using a Cell Counting Kit-8 assay, inhibited migration and invasion as determined using a wound healing assay or Transwell assay, and lamellipodia formation by filamentous actin staining, induced autophagy as determined using electron microscopy, and light chain 3B (LC-3B) expression, and apoptosis as determined using Annexin V staining in the two cell lines (P<0.05). Beclin 1 induced G arrest of HCT-15 transfectants as determined using propidium iodide staining (P<0.05), whereas HCT-116 transfectants were arrested in G phase (P<0.05). The two transfectants exhibited increased expression of c-Myc, cyclin D1, β-catenin, insulin-response element 1 and 78 kDa glucose-regulated protein compared with the control and mock cells as determined using the reverse transcription-quantitative polymerase chain reaction (P<0.05). Beclin 1 overexpression upregulated LC-3B and cyclin-dependent kinase 4 expression, but downregulated cyclin E expression of the cancer cell lines as determined using western blot analysis (P<0.05). Beclin 1 expression significantly suppressed the proliferation of colon cancer cells in xenograft models via inducing apoptosis by TUNEL, and inhibiting proliferation by Ki-67 expression (P<0.05). Beclin 1 overexpression may reverse aggressive phenotypes and suppress colon cancer tumor growth, and be employed as a target molecule for gene therapy of patients with colon cancer.
Beclin 1参与自噬、分化、凋亡及癌症进展，作为一个单倍体不足的肿瘤抑制基因发挥作用。本研究旨在阐明Beclin 1在结肠癌中的功能。将表达Beclin 1的质粒转染至HCT-15和HCT-116细胞中，并测定其表型及相关分子。将Beclin 1转染细胞皮下注射到裸鼠体内，分别使用Ki-67免疫染色和末端脱氧核苷酸转移酶介导的dUTP缺口末端标记法（TUNEL）来测定肿瘤生长、增殖及凋亡水平。使用细胞计数试剂盒8检测法测定，Beclin 1过表达抑制细胞活力；使用伤口愈合试验或Transwell试验测定，其抑制迁移和侵袭，并通过丝状肌动蛋白染色观察片状伪足形成；使用电子显微镜、轻链3B（LC-3B）表达检测，其诱导自噬；使用膜联蛋白V染色检测，其诱导凋亡，在两种细胞系中差异均有统计学意义（P<0.05）。使用碘化丙啶染色测定，Beclin 1诱导HCT-15转染细胞G期阻滞（P<0.05），而HCT-116转染细胞阻滞于G2期（P<0.05）。使用逆转录-定量聚合酶链反应测定，与对照细胞和空载体转染细胞相比，两种转染细胞中c-Myc、细胞周期蛋白D1、β-连环蛋白、胰岛素反应元件1和78 kDa葡萄糖调节蛋白的表达均增加（P<0.05）。使用蛋白质印迹分析测定，Beclin 1过表达上调癌细胞系中LC-3B和细胞周期蛋白依赖性激酶4的表达，但下调细胞周期蛋白E的表达（P<0.05）。在异种移植模型中，Beclin 1表达通过TUNEL法诱导凋亡及通过Ki-67表达抑制增殖，显著抑制结肠癌细胞的增殖（P<0.05）。Beclin 1过表达可能逆转侵袭性表型并抑制结肠癌肿瘤生长，可作为结肠癌患者基因治疗的靶分子。
