TCRP1基因在肺癌恶性转化中的作用及机制研究

TCRP1, known as tongue caner resistance-associated gene, was firstly cloned in our research group. Recently, we found TCRP1 is transcriptionally regulated by c-myc and TCRP1 expression in lung cancer tissues is much higher than that in normal lung tissues at both mRNA and protein levels. Moreover, TCRP1 maybe bind to Akt to enhance Akt activation to promote the malignant transformation of NIH3T3 cells in vitro. To further explore the role and mechanism of TCRP1 in the malignant transformation, proliferation and survival of lung cancer, in the present project, we will first investigate the changes of phenotype and related molecular in the TCRP1 and c-myc cotransfected normal bronchial epithelial cells. Then, we will observe the changes of malignant phenotype and molecular in lung cancer cells after TCRP1 or Akt knockdown. The third, we will construct the mutant-TCRP1 with Akt binding motif deletion and investigate the role of mutant-TCRP1 in the malignant transformation of NIH3T3 cells. The fourth, we will analysis the correlation between TCRP1 expression and its promoter methylation, c-myc binding site mutation and clinical characteristics including clinical stage, pathological type, metastasis and prognosis of lung cancer. Through these researches, we aim to clarify the oncogenic role of TCRP1 and provide new clues for the pathogenesis study of lung cancer.

TCRP1是本项目组自主克隆的舌癌耐药相关基因。前期研究发现：①TCRP1受c-myc转录激活，在肺癌mRNA和蛋白表达明显高于对照组织；②TCRP1通过Akt磷酸化激活而促NIH3T3细胞体外恶性转化；③TCRP1能与Akt在肺癌细胞结合并促进其磷酸化而激活下游信号分子。为深入探讨TCRP1在肺癌恶性转化、生长增殖及存活中的作用及调控机制，本项目拟首先采用TCRP1与c-myc共转染正常支气管细胞，观察表型和分子变化；其次通过干扰TCRP1、Akt在肺癌细胞的表达观察其恶性表型和分子变化；第三构建Akt结合区缺失的TCRP1突变载体，以野生型为对照转化NIH3T3细胞以识别TCRP1功能活性区；第四通过肺癌组织芯片检测分析TCRP1表达与启动子甲基化、c-myc结合区突变、肺癌临床分期、病理分型、转移及预后的相关关系。上述研究可望阐述TCRP1的癌基因样作用，为肺癌发病机制提供新的证据。

TCRP1 是本项目组自主克隆的舌癌耐药相关基因。本课题综合运用分子生物学、细胞生物学的多种研究手段在多种肺癌细胞系及小鼠模型中研究TCRP1在细胞转化及肺癌细胞恶性表型维持中的作用机制及其临床意义。结果发现：1）TCRP1 能与PDK1结合促进AKT磷酸化而激活下游信号分子，进而促进NIH3T3 细胞体外恶性转化；2）TCRP1在肺癌细胞、组织中高表达，且TCRP1表达与肺癌临床分期、淋巴结浸润相关及肺癌患者预后相关；3）体内、外实验分析发现敲除TCRP1 能够抑制肺癌细胞细胞增殖、克隆形成、诱导细胞周期阻滞，以及抑制裸鼠体内成瘤能力及移植瘤生长；3）TCRP1一方面通过AKT依赖的方式促进FOXO3a的磷酸化，另一方面通过SKP1促进FOXO3a的泛素化降解，进而抑制FOXO3a的表达而参与调控细胞周期进展。4）我们发现TCRP1受p53的调控，野生型p53直接结合并募集TET2和HDAC2至TCRP1启动子，下调TCRP1启动子活性。综上所述，本研究明确了TCRP1在细胞转化及肺癌细胞恶性表型维持中的作用并解析相关作用机制，为肺癌发病机制提供新的证据，并为以TCRP1 作为肺癌防治新靶点的抗肿瘤策略提供了实验基础。