CILP1通过激活KRAS通路促进结直肠癌恶性表型

CILP1 promotes the malignant phenotypes of colorectal cancer by activating the KRAS pathway

为探究软骨间层蛋白1(cartilage interlayer protein 1,CILP1)在结直肠癌发生、发展中的作用,采用生物信息学分析CILP1表达与结直肠癌分期、预后及Kirsten大鼠肉瘤病毒致癌基因(Kirsten rat sarcoma viral oncogene,KRAS)通路表达的关系;培养结直肠癌细胞SW620和HCT116,分为空载(Vector)组、 CILP1组(转染pcDNA 3.1-CILP1)以及对照组、 KRAS抑制剂Salirasib(Salirasib)组、 CILP1组、 CILP1+Salirasib组2套组别,采用CCK-8试验、 Transwell小室试验、 FACS检测细胞增殖、迁移、侵袭及凋亡;构建结直肠癌移植瘤模型,观察CILP1过表达对肿瘤生长的影响。结果显示,T3+T4分期、 N+分期、临床分期Ⅲ+Ⅳ患者CILP1表达水平分别高于T1+T2分期、 N0分期、临床分期Ⅰ+Ⅱ患者(P<0.05),CILP1低表达组患者总生存期(overall survival,OS)和无病生存期(disease-free survival,DFS)均高于CILP1高表达组(P<0.05)。与Vector组比较,CILP1组细胞增殖能力、迁移和侵袭细胞数及肿瘤体积、质量、 CILP1阳性表达数显著增加(P<0.05),细胞凋亡率显著降低(P<0.001)。基因集富集分析(gene set enrichment analysis,GSEA)显示,KRAS通路在CILP1高表达组显著上调(P<0.05)。与对照组比较,CILP1组CILP1、磷酸化丝氨酸/苏氨酸蛋白激酶1(phosphorylated serine/threonine protein kinase 1,p-Raf1)、磷酸化丝裂原激活蛋白和细胞外活化蛋白激酶(phosphorylated mitogen-activated protein/extracellular signal-regulated kinase,p-MEK)显著增加(P<0.05),Salirasib组CILP1、 p-Raf1、 p-MEK显著降低(P<0.05);与CILP1组比较,CILP1+Salirasib组CILP1、 p-Raf1、 p-MEK显著降低(P<0.001)。该研究提示,CILP1可能通过激活KRAS通路促进结直肠癌的发生、发展。

The aim of this study is to explore the roles of cartilage interlayer protein 1(CILP1) in the occurrence and development of colorectal cancer.The correlation of CILP1 expression and colorectal cancer stages,prognosis,and the Kirsten rat sarcoma viral oncogene(KRAS) pathway was analyzed by bioinformatics.The colorectal cancer cells SW620 and HCT116 were cultured and divided into 2 sets of groups(empty [Vector] group and CILP1 group [transfected with pcDNA 3.1-CILP1];control group,KRAS inhibitor Salirasib [Salirasib] group,CILP1 group,and CILP1+Salirasib group).Cell proliferation,migration,invasion,and apoptosis were detected by CCK-8 assay,Transwell chamber assay,and FACS,respectively.The xenografted tumor model of colorectal cancer was established to observe the effects of CILP1 overexpression on tumor growth.The results showed that the expressions of CILP1 in patients with stages T3+T4,N+,and clinical stages Ⅲ+Ⅳwere higher than those with stages T1+T2,N0,and clinical stages Ⅰ+Ⅱ(P<0.05).The overall survival(OS) and the disease-free survival(DFS) in the low-expression CILP1 group were higher than those in the high-expression CILP1 group(P<0.05).Compared to those of the Vector group,the proliferation,migration,and invasion of tumor cells,volume and mass of tumors in the xenografted mouse model,and positive expression of CILP1 were significantly increased in the CILP1 group(P<0.05),while the apoptosis rate of tumor cells was significantly decreased(P<0.001).Gene set enrichment analysis(GSEA) showed that the KRAS pathway was significantly up-regulated in the high-expression CILP1 group(P<0.05).Compared to those of the control group,the expressions of CILP1,phosphorylated serine/threonine protein kinase 1(p-Raf1),and phosphorylated mitogen-activated protein/extracellular signal-regulated kinase(p-MEK) were significantly increased in the CILP1 group(P<0.05),while significantly decreased in the Salirasib group(P<0.05).Compared to those of the CILP1 group,the expressions of CILP1,p-Raf1,and p-MEK were significantly decreased in the CILP1+Salirasib group(P<0.001).The above results suggest that CILP1 may promote the occurrence and development of colorectal cancer by activating KRAS pathway.