DNAJB6b通过激活AKT通路增强结直肠癌细胞的侵袭迁移能力

doi:10.3969/j.issn.1004-616x.2022.03.003

癌变·畸变·突变 ›› 2022, Vol. 34 ›› Issue (3): 178-183.doi: 10.3969/j.issn.1004-616x.2022.03.003

DNAJB6b通过激活AKT通路增强结直肠癌细胞的侵袭迁移能力

陈丁雄¹, 朱依青¹, 史建红¹, 蔡岩¹, 郝佳洁¹, 王明荣¹, 梁建伟², 张钰¹

1. 国家癌症中心/国家肿瘤临床医学研究中心/中国医学科学院北京协和医学院肿瘤医院分子肿瘤学国家重点实验室, 北京 100021;
2. 国家癌症中心/国家肿瘤临床医学研究中心/中国医学科学院北京协和医学院肿瘤医院结直肠外科, 北京 100021

收稿日期:2022-03-15 修回日期:2022-04-26 发布日期:2022-06-10
通讯作者: 梁建伟, 张钰
作者简介:陈丁雄,E-mail:cdxyzznl@163.com
基金资助:
国家自然科学基金(82073093)

DNAJB6b enhanced invasion and migration abilities of colorectal cancer cells by activating the AKT pathway

CHEN Dingxiong¹, ZHU Yiqing¹, SHI Jianhong¹, CAI Yan¹, HAO Jiajie¹, WANG Mingrong¹, LIANG Jianwei², ZHANG Yu¹

1. State Key Laboratory of Molecular Oncology, National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100021;
2. Department of Colorectal Surgery, National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100021, China

Received:2022-03-15 Revised:2022-04-26 Published:2022-06-10

摘要/Abstract

摘要： 目的: 探讨DnaJ热休克家族成员B6异构体b(DNAJB6b)过表达对结直肠癌细胞侵袭迁移能力的影响及相关分子机制。方法: 使用GEO数据库中的数据统计并分析结直肠癌组织中DNAJB6a和DNAJB6b mRNA表达水平的改变;体外培养结直肠癌细胞系DLD-1和HCT116,分别使用小干扰RNA(siRNA)和短发卡RNA(shRNA)敲降DNAJB6b的表达,以转染阴性对照siRNA和shRNA的细胞作为对照,使用Western blot检测敲降组与对照组细胞中相关蛋白表达水平的变化;使用PI3K/mTOR双重抑制剂BEZ235处理DLD-1和HCT116细胞,以溶剂处理细胞作为对照,进行Transwell侵袭和迁移实验,统计穿膜细胞数目;在稳定敲降DNAJB6b的DLD-1和HCT116细胞中,瞬时转染组成型活化的AKT(myr-AKT)表达载体,进行挽救实验,以转染相应空载体的细胞作为对照,使用Western blot检测相关蛋白的表达水平,同时进行Transwell实验评估细胞侵袭迁移能力的变化。结果: 与癌旁正常组织相比,结直肠癌组织中DNAJB6b mRNA的表达水平显著上调(P<0.05),而DNAJB6a mRNA的表达水平无显著变化。与对照组相比,在结直肠癌细胞系DLD-1和HCT116中敲降DNAJB6b的表达,可明显降低p-AKT(Ser473)的蛋白水平;BEZ235处理组细胞穿膜数目显著减少,仅为对照组的20%左右(P<0.01)。挽救实验结果显示,在稳定敲降DNAJB6b的DLD-1和HCT116细胞中过表达外源myr-AKT,与对照组相比,p-AKT(Ser473)表达水平升高并可显著逆转由于敲降DNAJB6b表达导致的细胞穿膜数目降低(P<0.01)。结论: DNAJB6b在结直肠癌组织中表达上调,其过表达可通过激活AKT通路增强结直肠癌细胞的侵袭迁移能力,提示其异常表达在结直肠癌发生进展过程中发挥促癌作用。

关键词: 结直肠癌, DNAJB6b, 侵袭迁移, AKT, 促癌作用

Abstract: OBJECTIVE: To investigate effects from over-expression of DnaJ heat shock protein family member B6 isoform b (DNAJB6b) on invasive and migration abilities of colorectal cancer (CRC) cells. METHODS: Expressions of DNAJB6b in cultured CRC cell lines DLD-1 and HCT116 were knocked down with small interfering RNA (siRNA) and short hairpin RNA (shRNA),respectively.Control cells were transfected with negative control siRNA and shRNA.Changes in protein levels were detected by Western blot.Changes of DNAJB6a and DNAJB6b mRNA levels in CRC specimens were statistically analyzed using the GEO dataset.DLD-1 and HCT116 cells were treated with PI3K/mTOR dual inhibitor BEZ235 and control cells were treated with solvents.Then,transwell invasion and migration assays were performed and the number of transmembrane cells were counted.Constitutively activated AKT (myr-Akt) was transiently-transfected into DNAJB6b stably-depleted DLD-1 and HCT116 cells (Referred as DLD-1-shD6b,HCT116-shD6b in this paper) and the controls were transfected with the empty vector.The rescue assay were conducted,changes in protein expression levels were detected by Western blot,and levels of cell invasion and migration were evaluated by Transwell assays. RESULTS: Compared with adjacent normal tissues,mRNA expressions of DNAJB6b but not DNAJB6a were significantly up-regulated in the CRC tissues (P<0.05).DNAJB6b depletion markedly reduced the p-Akt (Ser473) protein levels in DLD-1 and HCT116 cells compared with the control groups.The numbers of transmembrane cells treated by BEZ235 were significantly reduced compared to only about 20% of the control group (P<0.01).Resultsfrom the rescue assay showed that enforced expression of exogenous myr-Akt in DLD-1-shD6b and HCT116-shD6b cells up-regulated the p-Akt (Ser473) levels and prominently reversed the decreased number of transmembrane cells caused by DNAJB6b knockdown (P<0.01). CONCLUSION: DNAJB6b was up-regulated in CRC tissues,and its overexpression enhanced the invasion and migration abilities of CRC cells by activating the AKT pathway.Our observations suggest that these abnormal expressions played an oncogenic role in the development of CRC.

Key words: colorectal cancer, DNAJB6b, invasion and migration, AKT, oncogenic role

中图分类号:

R73-37

陈丁雄, 朱依青, 史建红, 蔡岩, 郝佳洁, 王明荣, 梁建伟, 张钰. DNAJB6b通过激活AKT通路增强结直肠癌细胞的侵袭迁移能力[J]. 癌变·畸变·突变, 2022, 34(3): 178-183.

CHEN Dingxiong, ZHU Yiqing, SHI Jianhong, CAI Yan, HAO Jiajie, WANG Mingrong, LIANG Jianwei, ZHANG Yu. DNAJB6b enhanced invasion and migration abilities of colorectal cancer cells by activating the AKT pathway[J]. Carcinogenesis, Teratogenesis & Mutagenesis, 2022, 34(3): 178-183.

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DNAJB6b通过激活AKT通路增强结直肠癌细胞的侵袭迁移能力

DNAJB6b enhanced invasion and migration abilities of colorectal cancer cells by activating the AKT pathway

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