CSMD3通过调控SKP2表达促进食管鳞癌细胞异常增殖的分子机制研究

Esophageal squamous cell carcinoma (ESCC) is a common malignant tumor with high incidence and high mortality in China. Due to the lack of effective diagnosis and treatment, the prognosis of ESCC is poor. CSMD3 is a new susceptible gene sequenced from 104 ECSS cases. The expression of CSMD3 was negatively correlated with the prognosis in other tumors. Previous experiments showed that the expression of CSMD3 in ESCC tissue was significantly higher than that in the adjacent tissues. After knocking out of CSMD3, the proliferation of ESCC was significantly inhibited, and the cells was arrested at G1 stage. The results of Cancer Pathway Finder PCR Array showed that gene SKP2 was significantly down regulated with CSMD3 at the same time. The abnormal expression of SKP2 could shorten the normal cell cycle time and promote abnormal proliferation. The results mentioned above indicated that CSMD3 may regulate SKP2 and its downstream target genes to promote the abnormal proliferation of ESCC. Therefore, the project for further study of tumor formation and transferring in vivo will be done through injecting the ESCC cells in model animals. And the immunoprecipitation, protein mass spectrometry and siRNA/ over-expression will be used to detect whether the CSMD3 signal pathway works through Skp2 and its downstream genes. The study will facilitate prevention, prognosis and treatment of ESCC.

食管鳞癌是我国高发、高致死的常见恶性肿瘤，由于缺乏有效的诊断和治疗方法，预后极差。CSMD3基因是课题组前期对104例食管鳞癌患者基因测序新发现的易感基因。生物信息学分析其他肿瘤中CSMD3的表达与预后呈负相关。初步实验表明，食管癌组织中CSMD3表达量显著高于癌旁组织；干扰CSMD3，可显著抑制细胞增殖，将细胞阻滞于G1期，PCR array表明基因SKP2同时显著下调。SKP2异常表达可使细胞快速从G1期进入S期、缩短正常细胞周期时间，达到异常增殖结果。我们推测，CSMD3调控SKP2及其下游靶基因促进食管癌细胞异常增殖。因此，本项目拟采用裸鼠皮下成瘤及尾静脉肿瘤转移模型在动物水平验证CSMD3促进食管癌肿瘤生长，转移；采用免疫共沉淀、蛋白质谱分析、siRNA/基因过表达等技术明确CSMD3在食管鳞癌中是否通过SKP2发挥癌基因作用。本研究将为有效防治食管癌提供新思路和药物作用新靶点。

食管鳞癌(ESCC)是我国高发、高致死的恶性肿瘤，由于缺乏有效的诊断和治疗方法，导致其预后不佳。因此，探讨食管癌发病的分子机制、提高早诊早治的准确度具有重要意义。CSMD3基因是课题组前期鉴定出的与食管鳞癌相关的高频突变基因。前期实验结果提示CSMD3在ESCC中起癌基因作用，其表达量与患者预后效果相反。但是CSMD3基因在ESCC中的具体作用机制尚未明确。因此，本项目利用多种生物学研究方法，从分子、蛋白、细胞水平对其在ESCC中可能的机制进行研究。实验结果如下：（1）利用慢病毒感染ESCC细胞以敲低CSMD3基因，发现S期激酶相关蛋白Skp2表达量随之显著下降，激光共聚焦实验表明CSMD3与Skp2在细胞内的表达位置存在重合，提示二者之间可能存在互作；（2）CSMD3表达不变，单独敲低Skp2基因，ESCC细胞增殖、侵袭、体外克隆以及裸鼠皮下肿瘤形成等能力受到显著抑制，细胞凋亡率升高，细胞被阻滞在G1期无法继续增殖，提示CSMD3可能通过Skp2发挥作用；（3）进一步研究其分子机制，当敲低Skp2基因时，P21、P27、P53、P300的表达量均显著升高，而拯救实验后，P21、P27、P53、P300的表达量均显著降低，同时，与细胞侵袭、迁移等高度相关的细胞骨架蛋白在Skp2基因敲低前后均有显著变化。综上，实验结果提示CSMD3基因通过调控Skp2基因的表达，进而调控与细胞周期进程相关的 p27和 p21的表达，促进 ESCC细胞的增殖；通过调节细胞骨架蛋白的表达，促进肿瘤细胞的侵袭、迁移；通过调节P53-P300信号通路，抑制细胞的凋亡，进而发挥癌基因的作用。本研究旨在明确CSMD3基因在ESCC中的作用，并研究其下游靶基因Skp2在ESCC中的作用及机制，为改善食管鳞癌患者的预后、寻找抗癌药物新靶点提供基础依据。