天津医药

天津医药 ›› 2019, Vol. 47 ›› Issue (5): 454-458.doi: 10.11958/20182153

• 细胞与分子生物学 • 上一篇    下一篇

PDCD4表达下调通过pAKT/pGSK3β途径促进胃癌顺铂耐药的机制初探

柳丹 1，汤志明 1，武福云 1，赵红艳 1，柯镜 1，向鹏 3，金佳晴 4，李珊 1，2△   

  1. 基金项目：国家自然科学基金资助项目（81502637，81702639）；湖北省科技厅知识创新专项（自然科学基金）（2018CFB467，2015CFB240）； 湖北省卫生计生委科研项目（WJ2019M052，WJ2019F065） 作者单位：1湖北医药学院基础医学研究所（邮编442000）；2湖北医药学院附属东风医院综合医疗科；3射阳县人民医院骨科；4武汉市第 八医院检验科 作者简介：柳丹（1993），女，在读硕士研究生，主要从事肿瘤耐药机制研究 △通讯作者 E-mail：lishanhbmu@126.com
  • 收稿日期:2018-12-29 修回日期:2019-03-15 出版日期:2019-05-15 发布日期:2019-05-15
  • 通讯作者: 李珊 E-mail:34141003@qq.com
  • 基金资助:
    PDCD4-Akt反馈环路在胃癌顺铂耐药中的作用机制研究

The molecular mechanism of PDCD4 knockdown facilitating cisplatin resistance in gastric cancer through pAKT/pGSK3β pathway

LIU Dan1, TANG Zhi-ming1, WU Fu-yun1, ZHAO Hong-yan1, KE Jing1, XIANG Peng3, JIN Jia-qing4, LI Shan1, 2△   

  1. 1 Institute of Basic Medical Sciences, Hubei University of Medicine, Shiyan 442000, China; 2 Department of Integrated Medicine, General Hospital of Dongfeng, Hubei University of Medicine; 3 Department of Orthopedics, People’s Hospital of Sheyang County; 4 Department of Clinical Laboratory, Wuhan Eighth Hospital △Corresponding Author E-mail: lishanhbmu@126.com
  • Received:2018-12-29 Revised:2019-03-15 Published:2019-05-15 Online:2019-05-15

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摘要： 摘要：目的 探讨程序性细胞凋亡因子4（PDCD4）表达下调在顺铂诱导胃癌细胞凋亡中的作用机制，为胃癌的 顺铂耐药提供新的分子靶点。方法 通过在人胃癌细胞系SGC7901中敲减表达PDCD4基因，顺铂诱导细胞凋亡，将 细胞分为shNC组、shPDCD4组、shNC加药组和shPDCD4加药组，荧光实时定量PCR（qRT-PCR）检测PDCD4 mRNA 表达水平；Caspase-3活性测定试剂盒、Hoechst染色结合细胞免疫荧光检测细胞凋亡水平；Western blot检测磷酸化蛋 白激酶B（pAKT）和磷酸化糖原合成酶激酶3β（pGSK3β）蛋白表达水平。在细胞中加入特异性Akt抑制剂，将细胞分 为 shNC 加药组、shPDCD4 加药组、LY294002 阻断组（转染 shPDCD4 质粒，先加 LY294002 再加入顺铂处理）和 Wortmannin 阻断组（转染 shPDCD4 质粒，先加 Wortmannin 再加入顺铂处理），Western blot 检测聚 ADP-核糖聚合酶 （PARP）蛋白表达水平。结果 qRT-PCR 和Western blot 检测结果证实稳定转染ShPDCD4质粒的SGC7901细胞中 PDCD4的mRNA表达和蛋白表达均有显著下降，体外稳定转染ShPDCD4的SGC7901胃癌细胞株构建成功。相较于 shNC 加药组，shPDCD4 加药组 Caspase3 活性下降，细胞凋亡降低（P＜0.05）。PDCD4 表达下调将导致 pAKT 和 pGSK3β的表达水平提高（P＜0.05），应用Akt抑制剂（LY294002、Wortmannin）阻断该信号通路后，PARP的相对表达 水平重新上调（P＜0.05）。结论 PDCD4表达下调能够通过pAKT/pGSK3β途径降低顺铂引起的胃癌细胞凋亡，进而 促进细胞顺铂耐药的形成。

关键词: 顺铂, 抗药性, 肿瘤, 胃肿瘤, 程序性细胞凋亡因子4, 蛋白激酶B, 糖原合成酶激酶3β

Abstract: Abstract: Objective To investigate the role of programmed apoptotic factor 4 (PDCD4) deficiency in the cisplatin induced apoptosis, and provide the new target marker for cisplatin resistance in gastric cancer. Methods ShNC/shPDCD4 vector was stably transfected into human gastric cancer cell line SGC7901 with or without cisplatin. The cells were divided into shNC group, shPDCD4 group, shNC with cisplatin group, shPDCD4 with cisplatin group for the following experiments. Real-time PCR and Western blot assay were used to detect the mRNA and protein expression levels of PDCD4. The caspase-3 activity kit and Hoechest dying with immunofluorescence were used to measure the cell apoptosis in vitro. The protein levels of pAKT and p-GSK3β were detected by Western blot assay. After combined with Akt inhibitor, the cells were divided into shNC with cisplatin group, shPDCD4 with cisplatin group, LY294002 inhibitor group and Wortmannin inhibitor group, and the protein levels of PARP were detected by Western blot assay. Results The results of qRT-PCR and Western blot assay confirmed that the mRNA and protein expressions of PDCD4 decreased significantly in SGC7901 cells, which stably transfected with ShPDCD4 plasmid, decreased significantly. The SGC7901 gastric cancer cell line that stably transfected with ShPDCD4 was successfully constructed in vitro. Caspase-3 activity and apoptosis were significantly decreased in shPDCD4 with cisplatin group than those of shNC with cisplatin group (P＜0.05). The expressions of pAKT and pGSK3β increased due to the deficiency of PDCD4 expression (P＜0.05). After blocking the signal pathway with Akt inhibitors (LY294002 and Wortmannin), the relative expression level of PARP was up-regulated (P＜0.05). Conclusion The down-regulation of PDCD4 expression can reduce the apoptosis of gastric cancer cells induced by cisplatin through pAKT/pGSK3 beta pathway, thus promoting the formation of cisplatin resistance.

Key words: cisplatin, drug resistance, neoplasm, stomach neoplasms, programmed apoptosis factor 4, protein kinase B, glycogen synthase kinase 3β