中国癌症杂志 ›› 2020, Vol. 30 ›› Issue (1): 25-31.doi: 10.19401/j.cnki.1007-3639.2020.01.003

• 论著 • 上一篇    下一篇

脂联素通过AMPK/mTOR/4EBP1通路对子宫内膜癌细胞迁移及侵袭的影响

隆曜先,王毛毛,蔡志福,张洁清,李 力   

  1. 广西医科大学附属肿瘤医院妇科,广西 南宁 530021
  • 出版日期:2020-01-30 发布日期:2020-01-17
  • 通信作者: 张洁清 E-mail: zjqmedical@163.com
  • 基金资助:
    广西自然科学基金(2015GXNSFAA139127);广西区域性高发肿瘤早期防治研究重点实验室(GK2015-ZZ08);北京医卫健康公益基金(YWJKJJHKYJJ-B17410-B06);广西壮族自治区临床重点专科建设项目。

Effect of adiponectin on migration and invasion of endometrial cancer cells via AMPK/mTOR/4EBP1 pathway

LONG Yaoxian, WANG Maomao, CAI Zhifu, ZHANG Jieqing, LI Li   

  1. Department of Gynecologic Oncology, Affiliated Tumor Hospital of Guangxi Medical University, Nanning 530021, Guangxi Zhuang Autonomous Region, China
  • Online:2020-01-30 Published:2020-01-17
  • Contact: ZHANG Jieqing E-mail:zjq201008@hotmail.com

摘要: 背景与目的:脂联素(adiponectin,APN)被认为是一种强有力的抑制肿瘤细胞生长的抗癌因子,但APN调节细胞转移的作用机制尚不清楚。探讨在子宫内膜癌HEC-1B细胞中,APN能否通过腺苷酸活化蛋白激酶(AMP-activated protein kinase,AMPK)/哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)/磷酸化真核启动因子4E结合蛋白1(4E binding protein 1,4EBP1)通路抑制细胞迁移及侵袭。方法:实验设计为4组:① APN组:20 μg/mL APN作用于细胞30 min;② 抑制剂组:10 μmol/L复合物C(AMPK抑制剂)作用于细胞30 min;③ APN+抑制剂组:10 μmol/L复合物C预处理细胞30 min后,加入20 μg/mL APN作用30 min;④ 对照组:仅加入无血清DMEM培养基。实时荧光定量聚合酶链反应(real-time fluorescence quantitative polymerase chain reaction,RTFQ-PCR)技术检测4组细胞中B细胞淋巴瘤-2(B- cell lymphoma 2,Bcl-2)和基质金属蛋白酶9(matrix metallopeptidase 9,MMP-9)的mRNA表达;蛋白质印迹法(Western blot)检测4组细胞中Bcl-2、MMP-9及AMPK信号通路中AMPK、mTOR、4EBP1蛋白水平;Transwell及划痕实验检测4组HEC-1B细胞的迁移能力。结果:Bcl-2和MMP-9 mRNA的表达水平在APN组中分别为0.64±0.08和0.68±0.02,均明显低于APN+抑制剂组(分别为0.98±0.11和0.96±0.08;P=0.02和0.03)。APN组中,HEC-1B细胞Bcl-2、MMP-9蛋白水平分别与对照组、抑制剂组、APN+抑制剂组比较,差异均有统计学意义(P=0.00)。APN组中,HEC-1B细胞AMPK、mTOR、4EBP1蛋白水平分别为1.49±0.02、0.48±0.00、0.19±0.00。与对照组比较,蛋白水平明显升高(P=0.00)。APN组穿膜细胞数为(78.72±10.74)个,APN+抑制剂组为(131.45±9.11)个,对照组为(131.91±12.29)个,APN组与对照组比较,差异有统计学意义(P=0.00),APN+抑制剂组与对照组相比,差异无统计学意义(P=0.12)。APN组中,HEC-1B细胞迁移率为(19.27±1.14)%,与其余3组比较,4组组间差异均有统计学意义(P=0.00),其余3组组间差异无统计学意义(F=2.78,P=0.39)。结论:APN可经AMPK/mTOR/4EBP1信号通路发挥抑制子宫内膜癌细胞迁移及侵袭的效应,达到抗肿瘤作用。

关键词:  子宫内膜癌, 脂联素, 腺苷酸活化蛋白激酶类, 4E结合蛋白1

Abstract: Background and purpose: Adiponectin (APN) is considered to be a potent anti-cancer factor that inhibits tumor cell growth, but the mechanism by which APN regulates cell metastasis remains unclear. This study investigated whether APN can pass AMP-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR)/phosphorylated eukaryotic promoter 4E binding protein 1 (4EBP1) in endometrial cancer HEC-1B cells to inhibit cell migration and invasion. Methods: There were 4 experimental groups: ① APN group: 20 μg/mL APN treated cells for 30 min; ② inhibitor group: 10 μmol/L complex C (AMPK inhibitor) treated cells for 30 min; ③ APN+inhibitor group: 10 μmol/L complex C pretreated cells for 30 min followed by incubation with 20 μg/mL APN for 30 min; ④ control group: only serum-free DMEM medium was added. The expression levels of B cell lymphoma-2 (Bcl-2) and matrix metallopeptidase 9 (MMP-9) mRNA in 4 groups of cells were detected by real-time fluorescence quantitative polymerase chain reaction (RTFQ-PCR). Western blot was used to detect the protein activation levels of AMPK, mTOR and 4EBP1 in Bcl-2, MMP-9 and AMPK signaling pathways in 4 groups of cells. Transwell and scratch assays were used to detect the migration ability of HEC-1B cells in four groups. Results: The expression levels of Bcl-2 and MMP-9 mRNA in the APN group were 0.64±0.08 and 0.68±0.02, respectively, which were significantly lower than those in the APN+inhibitor group (0.98±0.11 and 0.96±0.08; P=0.02 and 0.03). In the APN group, the protein expressions of Bcl-2 and MMP-9 in HEC-1B cells were significantly different from those in the control group, inhibitor group and APN+ inhibitor group (P=0.00). The activation levels of AMPK, mTOR and 4EBP1 proteins in HEC-1B cells of APN group were 1.49±0.02, 0.48±0.00 and 0.19±0.00, respectively. Protein levels were significantly enhanced compared with the control group (P=0.00). The number of transmembrane cells was 78.72±10.74 in the APN group, 131.45±9.11 in the APN+ inhibitor group, and 131.91±12.29 in the control group. The difference in the number of transmembrane cells between the APN group and the control group was statistically significant (P=0.00), while there was no significant difference between the APN+ inhibitor group and the control group (P=0.12). The mobility of HEC-1B cells in AP-N group was (19.27±1.60)%, which was significantly lower than that of the control group [(66.51±1.19)%]. The difference in the mobility of HEC-1B cells between the four groups was statistically significant (P=0.00), while there was no significant difference between the three groups (F=2.78, P=0.39). Conclusion: APN can inhibit the migration and invasion of endometrial cancer cells through AMPK/mTOR/4EBP1 signaling pathway and achieve its anti-tumor effect.

Key words: Endometrial neoplasms, Adiponectin, AMP-activated protein kinases, 4E binding protein 1