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:①APN组:20μg/mL APN作用于细胞30 min;②抑制剂组:10μmol/L复合物C(AMPK抑制剂)作用于细胞30 min;③APN+抑制剂组:10μmol/L复合物C预处理细胞30 min后,加入20μg/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。
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:①APN group:20μg/mL APN treated cells for 30 min;②inhibitor group:10μmol/L complex C(AMPK inhibitor)treated cells for 30 min;③APN+inhibitor group:10μmol/L complex C pretreated cells for 30 min followed by incubation with 20μg/mL APN for 30 min;④control 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.
作者
隆曜先
王毛毛
蔡志福
张洁清
李力
LONG Yaoxian;WANG Maomao;CAI Zhifu;ZHANG Jieqing;LI Li(Department of Gynecologic Oncology,Affiliated Tumor Hospital of Guangxi Medical University,Nanning 530021,Guangxi Zhuang Autonomous Region,China)
出处
《中国癌症杂志》
CAS
CSCD
北大核心
2020年第1期25-31,共7页
China Oncology
基金
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
