中国癌症杂志 ›› 2021, Vol. 31 ›› Issue (1): 35-44.doi: 10.19401/j.cnki.1007-3639.2021.01.005

• 论著 • 上一篇    下一篇

miR-122-5p通过靶向CREB1抑制食管癌细胞及移植瘤的生长

陈劭赓,何荣琦,张万飞,林贤钻,陈河山,许荣誉   

  1. 福建医科大学附属泉州第一医院胸外科,福建 泉州 362000
  • 出版日期:2021-01-30 发布日期:2021-02-20
  • 通信作者: 许荣誉 E-mail: xry641127@sina.com
  • 基金资助:
    福建省医学创新课题资助计划(2019-CX-46)。

miR-122-5p inhibits the growth of esophageal cancer cells and transplanted tumors by targeting CREB1

CHEN Shaogeng, HE Rongqi, ZHANG Wanfei, LIN Xianzuan, CHEN Heshan, XU Rongyu#br#   

  1. Department of Thoracic Surgery, Quanzhou First Hospital Affiliated to Fujian Medical University, Quanzhou 362000, Fujian Province, China
  • Published:2021-01-30 Online:2021-02-20
  • Contact: XU Rongyu E-mail: xry641127@sina.com

摘要: 背景与目的:miR-122在多种肿瘤中表达异常,参与肿瘤细胞增殖、凋亡等,而cAMP反应元件结合蛋白1(cAMP response element-binding protein 1,CREB1)参与食管癌发展过程,研究miR-122-5p通过靶向CREB1对食管癌细胞及移植瘤生长的作用及机制。方法:选取2017年11月—2019年11月于福建医科大学附属泉州第一医院行肿瘤切除术后保存在液氮中的食管癌及相应癌旁正常组织(距癌边缘3~5 cm)标本43例。采用实时荧光定量聚合酶链反应(real-time fluorescence quantitative polymerase chain reaction,RTFQ-PCR)检测食管癌组织和细胞中miR-122-5p mRNA和CREB1 mRNA的表达。将细胞分为对照组、miR-NC组、pc-NC组、miR-122-5p mimic组、pc-CREB1组、miR-122-5p mimic+pc-CREB1组,根据Lipofectamine TM 2000说明书将miR-NC、pc-NC、miR-122-5p mimic、pc-CREB1质粒分别或联合转染进入EC109细胞中。采用双萤光素酶报告基因实验分析靶向关系,采用MTT法检测细胞增殖情况,采用克隆形成实验检测细胞生长能力,采用流式细胞术检测细胞凋亡率,采用蛋白质印迹法(Western blot)检测Ki-67、增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)、活化胱天蛋白酶-3、Bax、Bcl-2、CREB1蛋白的相对表达水平。所有裸鼠分为4组:control组、miR-122-5p mimic组、pc-CREB1组、miR-122-5p mimic+pc-CREB1组。在裸鼠左腋下皮下注射各转染过的EC109细胞。30 d后处死裸鼠,完整取出皮下肿瘤,测定移植瘤体积和质量,采用免疫组织化学法检测Ki-67标记指数和胱天蛋白酶-3的表达水平。结果:miR-122-5p在食管癌组织和细胞中低表达,而CREB1在食管癌组织和细胞中高表达(P均<0.01)。miR-122-5p靶向下调CREB1表达。与对照组相比,miR-122-5p组食管癌细胞克隆数目减少,PCNA和Ki-67标记指数降低,细胞凋亡率增加,活化胱天蛋白酶-3和Bax蛋白表达上调,Bcl-2蛋白表达下调,食管癌EC109移植瘤体积和质量减小,Ki-67阳性细胞比率增加,活化胱天蛋白酶-3阳性细胞比率减少(P均<0.01)。过表达CREB1可逆转miR-122-5p对食管癌细胞和移植瘤增殖和凋亡的影响。结论:miR-122-5p通过靶向下调CREB1来抑制食管癌细胞增殖,并诱导细胞凋亡,从而抑制食管癌细胞和移植瘤的生长。

关键词: miR-122-5p, cAMP反应元件结合蛋白1, 食管癌, 增殖, 凋亡

Abstract: Background and purpose: miR-122 is abnormally expressed in various tumors and involved in tumor cell proliferation and apoptosis, while cAMP response element-binding protein 1 (CREB1) is involved in esophageal cancer development. This study aimed to investigate the effect of miR-122-5p on proliferation of esophageal cancer cells and xenografts by targeting CREB1 and its mechanism. Methods: Forty-three specimens of esophageal cancer and corresponding adjacent normal tissues (3-5 cm from the edge of cancer) were collected after tumor resection in Quanzhou First Hospital Affiliated to Fujian Medical University from November 2017 to November 2019. The expressions of miR-122-5p mRNA and CREB1 mRNA in esophageal cancer tissues and cells were detected by real-time fluorescence quantitative polymerase chain reaction (RTFQ-PCR). Cells were divided into control group, miR-NC group, pc-NC group, miR-122-5p mimic group, pc-CREB1 group and miR-122-5p mimic+pc-CREB1 group. The miR-NC, pc-NC, miR-122-5p mimic and pc-CREB1 plasmids were transfected into EC109 cells separately or jointly using Lipofectamine TM 2000. The targeting relationship was verified by the dual-luciferase reporter assay. The cell proliferation was detected by MTT assay. Cell growth ability was tested by cloning formation experiment. The rate of apoptosis was detected by flow cytometry. The Ki-67 labelling index, proliferating cell nuclear antigen (PCNA), activated caspase-3, Bax, Bcl-2 and CREB1 were detected by Western blot. All nude mice were divided into four groups: control group, miR-122-5p mimic group, pc-CREB1 group, miR-122-5p mimic+pc-CREB1 group. Transfected EC109 cells were injected subcutaneously in the left armpit of nude mice. After 30 days, the nude mice were sacrificed, the subcutaneous tumors were completely removed, and the volume and weight of the transplanted tumors were measured. The Ki-67 labelling index and caspase-3 were detected by immunohistochemistry. Results: miR-122-5p was expressed at a low level in esophageal cancer tissues and cells, while CREB1 was highly expressed in esophageal cancer tissues and cells (both P<0.01). miR-122-5p targeted down-regulation of CREB1 was observed. Compared with the control group, the number of esophageal cancer cell clones in the miR-122-5p group decreased, PCNA and Ki-67 labelling index were down- regulated, the apoptotic rate increased, activated caspase-3 and Bax protein expressions were up-regulated, Bcl-2 protein expression was down-regulated, the volume and weight of esophageal cancer EC109 transplanted tumor decreased, the ratio of Ki-67 positive cells increased, and the ratio of caspase-3 positive cells decreased (all P<0.01). Overexpression of CREB1 reversed the effects of miR-122-5p on proliferation and apoptosis of esophageal cancer cells and transplanted tumors. Conclusion: miR-122-5p can inhibit the proliferation of esophageal cancer cells and induce apoptosis by targeted down-regulation of CREB1, thereby inhibiting the growth of esophageal cancer cells and transplanted tumors.

Key words: miR-122-5p, cAMP response element-binding protein 1, Esophageal cancer, Proliferation, Apoptosis