南方医科大学学报

南方医科大学学报 ›› 2023, Vol. 43 ›› Issue (4): 585-589.doi: 10.12122/j.issn.1673-4254.2023.04.11

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NKD1可促进结肠癌细胞的葡萄糖吸收:基于激活YWHAE基因的转录活性

刘 迁，戴宇阳，于华裔，沈 颖，邓建忠，陆文斌，金建华   

  1. 江苏大学附属武进医院/徐州医科大学武进临床学院，肿瘤科，江苏 常州 213017；常州市分子诊断与肿瘤精准医学重点实验室/江苏大学武进分子诊断与肿瘤精准医学研究院，江苏 常州 213017
  • 出版日期:2023-04-20 发布日期:2023-05-16

NKD1 promotes glucose uptake in colon cancer cells by activating YWHAE transcription

LIU Qian, DAI Yuyang, YU Huayi, SHEN Ying, DENG Jianzhong, LU Wenbin, JIN Jianhua   

  1. Department of Oncology, Wujin Hospital Affiliated to Jiangsu University/Wujin Clinical College, Xuzhou Medical University, Changzhou 213017, China; Changzhou Key Laboratory of Molecular Diagnostics and Precision Cancer Medicine/ Wujin Institute of Molecular Diagnostics and Precision Cancer Medicine of Jiangsu University, Changzhou 213017, China
  • Online:2023-04-20 Published:2023-05-16

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摘要： 目的 通过研究NKD1与YWHAE调控关系，分析NKD1促进肿瘤细胞增殖的新作用机制。方法 实验分组：（1）对照组：转染pcDNA3.0质粒的HCT116细胞、转染NC-siRNA的SW620细胞、正常HCT116细胞、正常SW620细胞；（2）实验组：转染pcDNA3.0-NKD1 质粒的 HCT116 细胞、转染 NKD1 siRNA 的 SW620 细胞、HCT116-NKD1 细胞、SW620-nkd1-/-细胞、转染pcDNA3.0-YWHAE质粒的SW620-nkd1-/-细胞。采用Western blot及qRT-PCR实验检测结肠癌细胞中过表达或敲除NKD1对YWHAE的蛋白及mRNA水平变化的影响。采用染色质免疫共沉淀（ChIP）技术检测NKD1是否结合YWHAE基因启动子区。通过双荧光素酶报告基因实验分析NKD1对YWHAE基因启动子活性的调控作用。此外，采用免疫荧光实验分析NKD1与YWHAE相互作用情况。葡萄糖检测实验分析NKD1对肿瘤细胞吸收葡萄糖的调控作用。结果 与对照组相比，过表达（或敲除）NKD1不仅在蛋白水平增强（或降低）YWHAE的表达，还在mRNA水平增加（或降低）其表达（P<0.001）。ChIP实验显示NKD1蛋白能够结合YWHAE启动子序列，双荧光素酶报告基因实验表明在结肠癌细胞中过表达（或敲降）NKD1能显著增强（或降低）YWHAE启动子的转录活性（P<0.05）。此外，免疫荧光结果显示NKD1蛋白与YWHAE蛋白在结肠癌细胞内相互结合。葡萄糖水平分析实验表明，敲除NKD1明显降低结肠癌细胞对葡萄糖的吸收水平（P<0.01），而在敲除NKD1的细胞中过表达YWHAE，则细胞对葡萄糖吸收能力得到恢复（P<0.05）。结论 结肠癌细胞中NKD1蛋白通过激活YWHAE基因的转录活性，促进其表达，从而促进肿瘤细胞对葡萄糖的吸收。

关键词: NKD1；YWHAE；葡萄糖吸收；结肠癌

Abstract: Objective Bo investigate the regulatory relationship between NKD1 and YWHAE and the mechanism of NKD1 for promoting tumor cell proliferation. Methods HCT116 cells transfected with pcDNA3.0-NKD1 plasmid, SW620 cells transfected with NKD1 siRNA, HCT116 cells with stable NKD1 overexpression (HCT116-NKD1 cells), SW620 cells with nkd1knockout (SW620-nkd1-/- cells), and SW620-nkd1-/- cells transfected with pcDNA3.0-YWHAE plasmid were examined for changes in mRNA and protein expression levels of YWHAE using qRT-PCR and Western blotting. Chromatin immunoprecipitation (ChIP) assay was used to detect the binding of NKD1 to the promoter region of YWHAE gene. The regulatory effect of NKD1 on YWHAE gene promoter activity was analyzed by dual-luciferase reporter gene assay, and the interaction between NKD1 and YWHAE was analyzed with immunofluorescence assay. The regulatory effect of NKD1 on glucose uptake was examined in the tumor cells. Results In HCT116 cells, overexpression of NKD1 significantly enhanced the expression of YWHAE at both the mRNA and protein levels, while NKD1 knockout decreased its expression in SW620 cells (P<0.001). ChIP assay showed that NKD1 protein was capable of binding to the YWHAE promoter sequence; dual luciferase reporter gene assay showed that NKD1 overexpression (or knockdown) in the colon cancer cells significantly enhanced (or reduced) the transcriptional activity of YWHAE promoter (P<0.05). Immunofluorescence assay demonstrated the binding of NKD1 and YWHAE proteins in colon cancer cells. NKD1 knockout significantly reduced glucose uptake in colon cancer cells (P<0.01), while YWHAE overexpression restored the glucose uptake in NKD1-knockout cells (P<0.05). Conclusion NKD1 protein activates the transcriptional activity of YWHAE gene to promote glucose uptake in colon cancer cells.

Key words: NKD1; YWHAE; glucose absorption; colon cancer