结肠癌中ID1上调OCT4信号通路的机制研究  

The upregulation mechanism of OCT4 signaling by ID1 in colorectal cancer

作　　者：尚爽 宋佳玮 花芳 SHANG Shuang;SONG Jia-wei;HUA Fang(State Key Laboratory of Bioactive Substance and Function of Natural Medicines,Beijing Key Laboratory of New Drug Mechanisms and Pharmacological Evaluation Study(BZ0150),Institute of Meteria Medica,Chinese Academy of Medical Sciences and Peking Union Medical College,Beijing 100050,China)

机构地区：[1]中国医学科学院、北京协和医学院药物研究所,天然药物活性物质与功能国家重点实验室,新药作用机制研究与药效评价北京市重点实验室(BZ0150),北京100050

出　　处：《药学学报》2021年第7期1945-1952,共8页Acta Pharmaceutica Sinica

基　　金：国家自然科学基金资助项目(81973344,81903636).

摘　　要：DNA结合抑制蛋白-1(inhibitor of DNA binding 1,ID1)在结肠癌、肺癌和乳腺癌等多种肿瘤组织中异常高表达,并与肿瘤恶性进展及患者不良预后密切相关。已有研究发现,ID1在结肠癌中可以维持肿瘤细胞干细胞样特性,介导肿瘤干细胞耐药,但其分子生物学机制并未被完全阐明。通过免疫印迹、基因集富集分析、双荧光素酶报告基因检测及实时定量PCR,本研究发现ID1可从转录水平上调维持肿瘤细胞干性的关键转录因子八聚体结合蛋白(octamer binding transcription factor,OCT4)的蛋白表达及信号通路活性。利用体外成球实验,本研究证明过表达OCT4可逆转敲低ID1对干细胞成球能力的抑制作用。在进一步的机制研究中,利用JASPAR及GEPIA数据库,本研究预测并得到能够负向调控OCT4转录的转录抑制因子叉头蛋白D3(forkhead box D3,FOXD3)。通过免疫共沉淀、免疫荧光共聚焦及实时定量PCR等方法,本研究最终证明ID1通过与转录抑制因子FOXD3相互作用,抑制其活性从而上调OCT4的转录及信号通路活性。综上,本研究为结肠癌干细胞的分子调控机制提供了新的理论基础,研究中发现的ID1-FOXD3蛋白-蛋白相互作用为结肠癌治疗提供了新的潜在干预靶点。Inhibitor of DNA binding 1(ID1)has an aberrantly high expression in multiple cancer tissues,including colon cancer,lung cancer,breast cancer,and so on,which is closely related to cancer aggressiveness and poor clinical outcomes in cancer patients.It has been reported that ID1 maintains colorectal cancer cells(CRCs)stemness traits and contributes to the CRC drug resistance.While,the biological molecular mechanisms have not been fully elucidated.In this research,we found that ID1 upregulates octamer binding transcription factor(OCT4)protein level as well as OCT4 signaling pathway via Western blot,gene set enrichment analysis(GSEA),dual-luciferase reporter assay,and real-time PCR.Through the in vitro sphere formation assay,we found that overexpression of OCT4 reverses the inhibitory effect of knocking down ID1 on CRC sphere formation ability.With the help of JASPAR and GEPIA database,we predicted a novel transcriptional repressor—forkhead box D3(FOXD3)of OCT4.Finally,by using co-immunoprecipitation(Co-IP),confocal and real-time PCR,we demonstrated that ID1 interacts with FOXD3 to inhibit its transcriptional repression activity and therefore to upregulate OCT4 transcription and OCT4 signaling pathway.In conclusion,this study provides a new theoretical basis for the regulation mechanism of colon cancer stem cells,and the newly found protein-protein interaction of ID1-FOXD3 provides a potential drug target for the therapy of CRC.

关 键 词：DNA结合抑制蛋白-1 叉头蛋白D3 八聚体结合转录因子 结肠癌干细胞 转录活性 

分 类 号：R966[医药卫生—药理学]