【摘要】 目的：研究沉默分泌型磷蛋白1（secretory phosphoprotein 1,SPP1）表达对结直肠癌（colorectal cancer,CRC）细胞增殖、迁移与侵袭的影响，及可能的作用机制。方法：用基因表达谱互动分析（Gene Expression Profiling Interactive Analysis,GEPIA）数据库获取CRC中SPP1基因的表达情况；采用免疫组织化学法和蛋白质印迹法检测远端正常结直肠组织、癌旁组织、CRC组织、正常结直肠细胞及CRC细胞中SPP1蛋白的表达情况。采用慢病毒感染的方法将携带有特异性针对SPP1基因的sh RNA转入CRC细胞HT-29和HCT-116，敲低SPP1基因的表达水平。随后，分别采用CCK-8法、细胞集落形成实验及Transwell小室实验检测CRC细胞的增殖、迁移和侵袭能力，再用蛋白质印迹法检测HT-29和HCT-116细胞中AKT/糖原合成酶激酶3β（glycogen synthase kinase 3β,GSK3β）信号通路的活化以及上皮-间质转化（epithelial-mesenchymal transition,EMT）相关蛋白的表达情况。最后，通过小鼠成瘤实验检测敲低SPP1表达对HT-29细胞移植瘤生长和肺转移的影响。结果：GEPIA数据库分析结果显示，SPP1 m RNA在CRC患者中的表达水平均显著上调（P均<0.05），且与患者的不良预后有关；CRC组织及细胞中SPP1蛋白的表达水平显著升高（P均<0.001）。敲低SPP1表达后，CRC细胞的活力、集落形成能力、迁移能力及侵袭能力均显著降低（P均<0.001）；细胞中磷酸化AKT （phospho-AKT,pAKT）、p-GSK3β、Snail及波形蛋白（Vementin）的表达水平均显著降低（P均<0.001），而E-钙黏蛋白（E-cadherin）表达水平显著升高（P<0.001）。敲低SPP1表达后能抑制小鼠中HT-29细胞移植瘤的生长和肺转移的发生。结论：敲低SPP1表达能抑制CRC细胞的增殖、迁移与侵袭能力，其作用机制可能与减弱AKT/GSK3β信号通路的活性有关。更多还原

【Abstract】 Objective: To investigate the roles of secretory phosphoprotein 1(SPP1) in the progression of colorectal cancer(CRC) and the underlying mechanism.Methods: Gene Expression Profiling Interactive Analysis(GEPIA) database was used to obtain the expression of SPP1 gene in CRC. Immunohistochemistry analysis and Western blotting were used to detect the expression of SPP1 in distal normal colorectal tissues,adjacent tissues, CRC tissues, normal colorectal cell lines and CRC cell lines. The cell viability, colony formation, migration and invasion of CRC cells as well as the activation of AKT/glycogen synthase kinase 3β(GSK3β) signaling pathway and the expression of epithelial-mesenchymal transition(EMT)-related proteins in HT-29 cells and HCT-116 cells were detected by CCK-8 assay, colony formation assay, trranswell assay and Western blotting after SPP1 knockdown in vitro through lentiviral infection carrying shRNA against SPP1 gene. Tumor formation assay was used to detect the effect of SPP1 knockdown on the growth and lung metastasis of transplanted HT-29 tumor in vivo.Results: SPP1 expression was significantly increased in CRC tissues and cell lines(P <0.001) and was associated with poor prognosis of CRC patients according to GEPIA database analysis. The expression of SPP1 protein was significantly upregulated in CRC tissues and cells(P < 0.001). After knockdown of SPP1 expression, the cell viability, colony formation,migration and invasion of CRC cells were significantly decreased(P < 0.001), the expression of phosphorylated AKT(phospho-AKT, p-AKT), phosphorylated GSK3β(phospho-GSK3β, pGSK3β), Snail and Vementin were significantly decreased(P < 0.001), while E-cadherin expression was significantly increased(P < 0.001). Knockdown of SPP1 expression inhibited the growth and lung metastasis of HT-29 cell tumor xenografts in mice.Conclusion: SPP1 knockdown can inhibit the proliferation, migration and invasion of CRC cells, which may be related to the reduction of AKT/GSK3β signaling activity.更多还原