CDK16调节食管鳞癌细胞生长及增殖的分子机制研究

Esophageal squamous cell carcinoma is a high incidence tumor in China. Patients with esophageal squamous cell carcinoma have poor prognosis and high mortality, and 5-year survival rate is 17% to 20%. Cell cycle progression is mainly regulated by Cyclins, cyclin-dependent kinases (CDKs) and cyclin-dependent kinase inhibitor (CDKI). CDKs are positively regulated by Cyclins and negatively regulated by CDKI. Abnormal expression of CDKs may cause disorder of cell cycle progression and may contribute to tumorigenesis. The results of previous studies showed that CDK16 has high expression in human esophageal squamous cell carcinoma generally and has low expression in adjacent tissues. CDK16 has high expression in poorly differentiated tissues, has general expression in moderately differentiated tissues and has low expression in highly differentiated tissues of esophageal squamous cell carcinoma. Expression of CDK16 and tumor differentiation was negatively correlated. And we also found that expression of CDK16 and Ki-67 was positively correlated. But the molecular mechanisms of regulationof CDK16 on cell growth and proliferation of esophageal squamous cell carcinoma needs to be further clarified. We plan to use colony formation assay, MTT assay and animal experiments to research the impact of CDK16 on cell growth and proliferation of esophageal squamous cell carcinoma. We also plan to use Co-immunoprecipitation, two-dimensional gel electrophoresis and mass spectrometry to screen interaction proteins of CDK16 and to find the related signaling pathways CDK16 involved in. This research could elucidate the molecular mechanisms of the regulation of formation and development of esophageal squamous cell carcinoma by CDK16, and this research could also provide new evidence for the prevention and treatment of esophageal squamous cell carcinoma.

食管鳞癌是我国高发肿瘤，患者预后差，死亡率高，5年生存率为17%~20%。细胞周期紊乱与食管鳞癌发生发展密切相关。细胞周期由细胞周期素、细胞周期素依赖性激酶、细胞周期素依赖性激酶抑制蛋白共同调节。细胞周期素依赖性激酶(CDKs)异常表达会引起细胞周期紊乱而导致肿瘤发生。我们前期研究发现CDK16在食管鳞癌组织高表达，在癌旁组织低表达；CDK16在食管鳞癌的低分化组织表达较高，在高分化组织表达最低，CDK16表达与肿瘤分化程度负相关。但CDK16调节食管鳞癌细胞生长及增殖的分子机制有待进一步阐明。我们拟采用集落形成实验、MTT测定及动物实验等研究CDK16对食管鳞癌细胞生长及增殖的影响；采用免疫共沉淀、双向凝胶电泳、质谱分析等筛选相互作用蛋白，发现相关信号通路。该研究可阐明CDK16参与食管鳞癌发生发展的分子机制，为食管鳞癌的防治提供新的依据。

本项目严格按照项目内容、项目方案及年度研究计划，对CDK16调控食管鳞癌发病的功能进行了扎实的研究。.本课题组首先通过免疫组化检测CDK16和Ki67在75例食管鳞癌的癌/癌旁组织中的表达。结果表明，CDK16在食管鳞癌的癌组织及癌旁组织中表达阳性率的差异具有统计学意义，CDK16的表达与肿瘤分化程度、淋巴结转移及TNM分期显著相关。相关性分析显示，CDK16与肿瘤细胞增殖指标Ki67的表达呈显著正相关。.通过qRT-PCR及Western Blot方法检测了EC1、EC109、TE1、 KYSE150、KYSE450和KYSE510这6种食管鳞癌细胞及1种正常人食管上皮细胞HET-1A中CDK16的表达。结果显示CDK16的表达在食管鳞癌细胞中显著高于正常人食管上皮细胞。其中EC109及KYSE150中CDK16的表达相对较高，被选用于后续的实验。本课题组构建了CDK16的慢病毒干扰载体，包装病毒并感染EC109和KYSE150，通过Puromycin药筛得到稳定沉默CDK16的食管鳞癌细胞系。.利用建立好的稳定沉默CDK16的食管鳞癌细胞系，通过克隆形成实验、MTT实验、Brdu实验检测了敲减CDK16对食管鳞癌的细胞生长及增殖的影响，结果表明，敲减CDK16显著减缓细胞生长及增殖；通过划痕实验、Transwell实验、Transwell(基质胶)实验检测了敲减CDK16对食管鳞癌的细胞迁移及侵袭的影响，结果表明，敲减CDK16显著抑制细胞的迁移及侵袭；通过流式细胞术PI染色法检测了敲减CDK16对食管鳞癌细胞周期的影响，结果显示敲减CDK16可以将食管鳞癌细胞阻滞在细胞周期的G2/M期；通过流式细胞术结合Annexin V-APC单染法检测了敲减CDK16对食管鳞癌细胞凋亡的影响，结果显示，敲减CDK16可以导致食管鳞癌细胞的凋亡率显著升高。.另外课题组将稳定沉默CDK16的KYSE150细胞接种于裸鼠皮下，构建了裸鼠皮下移植瘤模型。结果显示，实验组瘤体的生长速度明显减慢，瘤体的平均质量显著低于对照组。这表明沉默CDK16可以有效抑制移植瘤的生长。.本课题组构建了带有3×Flag和TST两种纯化标签的CDK16真核表达载体，通过串联亲和纯化并结合质谱分析，筛选鉴定出食管鳞癌细胞内CDK16的相互作用蛋白。.