Jie Feng, Bo Qi, Ling Guo, Xiu-Feng Wei, Yu-Zhen Liu, Bao-Sheng Zhao, Department of Thoracic Surgery, The First Affiliated Hospital of Xinxiang Medical University, Weihui 453100, Henan Province, China.
World J Gastroenterol. 2017 Jun 21;23(23):4243-4251. doi: 10.3748/wjg.v23.i23.4243.
To explore the effect of miR-382 on esophageal squamous cell carcinoma (ESCC) and its possible molecular mechanism.
Eca109 cells derived from human ESCC and Het-1A cells derived from human normal esophageal epithelium were used. Lentivirus-mediated miR-382 was overexpressed in Eca109 cells. The effect of miR-382 on cell proliferation was evaluated by MTT and colony formation assay. For cell cycle analysis, cells were fixed and stained for 30 min with propidium iodide (PI) staining buffer containing 10 mg/mL PI and 100 mg/mL RNase A, and analyzed by BD FACSCalibur™ flow cytometer. For cell apoptosis assay, cells were stained with an Annexin V-FITC/PI Apoptosis Detection Kit according to the manufacturer's instructions and analyzed by a dual-laser flow cytometer. Cell invasion and migration abilities were determined through use of transwell chambers, non-coated or pre-coated with matrigel. Levels of proteins related to cell growth and migration were examined by western blotting.
Endogenous miR-382 was down-regulated in Eca109 cells compared with Het-1A. Introduction of miR-382 not only significantly inhibited proliferation and colony formation, but also arrested cell cycle at the G2/M phase, as well as promoted apoptosis and autophagy in Eca109 cells. Migration, invasion and epithelial-mesenchymal transition of Eca109 cells were suppressed by overexpressing miR-382. Western blotting results showed that miR-382 inhibited the phosphorylation of mTOR and 4E-BP1.
miR-382 functions as a tumor suppressor against ESCC development and metastasis, and could be considered as a potential drug source for the treatment of ESCC patients.
探讨 miR-382 对食管鳞状细胞癌（ESCC）的影响及其可能的分子机制。
使用人 ESCC 来源的 Eca109 细胞和人正常食管上皮来源的 Het-1A 细胞。通过慢病毒转染过表达 Eca109 细胞中的 miR-382。通过 MTT 和集落形成实验评估 miR-382 对细胞增殖的影响。对于细胞周期分析，用包含 10mg/ml PI 和 100mg/ml RNase A 的 PI 染色缓冲液固定和染色细胞 30min，并用 BD FACSCalibur™流式细胞仪进行分析。对于细胞凋亡分析，根据制造商的说明用 Annexin V-FITC/PI 凋亡检测试剂盒对细胞进行染色，并用双激光流式细胞仪进行分析。通过使用未涂覆或预先涂覆有基质胶的 Transwell 室来测定细胞侵袭和迁移能力。通过 Western blot 检测与细胞生长和迁移相关的蛋白水平。
与 Het-1A 相比，Eca109 细胞中内源性 miR-382 下调。引入 miR-382 不仅显著抑制了增殖和集落形成，而且还使 Eca109 细胞的细胞周期停滞在 G2/M 期，同时还促进了细胞凋亡和自噬。过表达 miR-382 抑制了 Eca109 细胞的迁移、侵袭和上皮-间充质转化。Western blot 结果表明，miR-382 抑制了 mTOR 和 4E-BP1 的磷酸化。
miR-382 作为 ESCC 发展和转移的肿瘤抑制因子发挥作用，可被视为治疗 ESCC 患者的潜在药物来源。
