SPP1对人结直肠癌细胞侵袭、转移及上皮间质化的影响

Study on the Expression Difference of SPP1 in Different CRC Cell Lines and the Regulation Mechanism of EMT

目的探究分泌磷蛋白1(SPP1)在不同结直肠癌细胞系中的表达情况及SPP1对结直肠癌细胞上皮间质化(Epithelial-Mesenchymal transition,EMT)影响的机制。方法通过Western blot检测SPP1在正常结肠上皮细胞株(NCM460)、原位结肠癌细胞株(HCT116)、原发性结直肠腺癌细胞株(HT29)、原位直肠腺癌细胞株(SW480)、淋巴结转移结肠癌细胞株(SW620)等不同结直肠癌细胞株中的表达。SPP1沉默慢病毒转染HCT116,SPP1过表达慢病毒转染NCM460,利用Transwell、划痕实验分析转染后细胞的侵袭、迁移能力。Western blot检测慢病毒转染后HCT116和NCM460细胞中EMT通路关键蛋白的表达。结果Western blot结果显示,与NCM460细胞相比,SPP1在HCT116、HT29、SW480、SW620细胞中均表达上调(P<0.01)。转染后两种细胞的Transwell侵袭实验和划痕实验结果显示,上调SPP1表达使细胞侵袭及迁移能力增强(P均<0.01),沉默SPP1表达使细胞侵袭及迁移能力降低(P均<0.01);Western blot结果显示,上调细胞的SPP1水平使snail1、N-cadherin、Vimentin等蛋白表达上调(P<0.05),E-cadherin蛋白表达下调(P<0.05),激活EMT通路;下调细胞SPP1水平使snail1、N-cadherin、Vimentin等蛋白表达下调(P<0.05),E-cadherin蛋白表达上调(P<0.05),抑制EMT通路。结论SPP1在结直肠癌细胞中表达上调,其表达水平影响NCM460、HCT116细胞的EMT及侵袭、迁移能力。

Objective To investigate the expression of phosphoprotein 1(SPP1)in different colorectal cancer cell lines and the mechanism of the effect of SPP1 on Epithelial-Mesenchymal transition(EMT)of colorectal cancer cells.Methods The expression of SPP1 in different cell lines was detected by Western blot.SPP1 silenced lentivirus was transfected into colon cancer cell line in situ(HCT116),while SPP1 overexpressed lentivirus was transfected into normal colon epithelial cell line(NCM460).Transwell and scratch test were used to analyze the invasion and migration ability of the transfected cells.Western blot was used to detect the expression of key proteins of EMT pathway in HCT116 and NCM460 cells after lentivirus transfection.Results Western blot results showed that SPP1 was up-regulated in HCT116,HT29,SW480,and SW620 cells compared with NCM460 cells(P<0.01).Transwell invasion assay and scratch test showed that up-regulation of SPP1 expression increased cell invasion and migration ability(P<0.01).Silencing SPP1 expression reduced cell invasion and migration ability(P<0.01).Western blot results showed that upregulation of SPP1 increased the expression of snail1 protein,N-cadherin protein and Vimentin protein(P<0.05),and down-regulated E-cadherin expression(P<0.05)and promoted activation of the Epithelial-Mesenchymal transition(EMT)pathway.Down-regulation of SPP1 level resulted in down-regulation of snail 1,N-cadherin and Vimentin(P<0.05).Vimentin protein expression was down-regulated(P<0.05)and EMT pathway was inhibited.Conclusion SPP1 plays an important role in the development of colorectal cancer.The expression level of SPP1 affects the epithelialization,invasion and migration of NCM460 and HCT116 cells.