敲减TIM-3对结直肠癌细胞生长及分泌免疫抑制分子的影响

Effects of TIM-3 knockdown on the growth and immunosuppressive molecules secretion of colorectal cancer cells

目的研究敲减T免疫球蛋白及黏蛋白分子3(TIM-3)对结直肠癌细胞生长及细胞分泌免疫抑制分子的影响。方法设置稳定感染TIM-3 shRNA和shRNA control重组慢病毒的SW620细胞株为sh-TIM-3和sh-NC,设置不添加慢病毒的SW620细胞为Control组,细胞培养2 d以后,qRT-PCR和Western blot检测细胞中TIM-3表达水平。MTT方法检测细胞增殖情况,平板克隆实验检测细胞克隆形成能力,PI单染法检测细胞周期,Western blot方法检测细胞中细胞周期蛋白D1(CyclinD1)、p27蛋白水平,ELISA法检测细胞分泌的血管内皮生长因子(VEGF)、白细胞介素-10(IL-10)水平。结果sh-TIM-3组细胞中的TIM-3 mRNA和蛋白表达水平均降低,细胞增殖能力降低,细胞克隆形成能力也降低,细胞G 0/G 1比例降低,CyclinD1蛋白水平降低,p27蛋白水平升高,细胞分泌的VEGF、IL-10减少,与Control组比较,差异具有统计学意义(P<0.05)。结论敲减TIM-3能够抑制结直肠癌细胞生长并减少细胞分泌免疫抑制因子。

To evaluate the effect of TIM-3 knockdown on the cell growth and immunosuppressive molecules secretion of colorectal cancer cells,the SW620 cell lines were stably infected with TIM-3 shRNA and shRNA control recombinant lentiviruses,namely sh-TIM-3 and sh-NC,while the SW620 cells without lentiviruses were set as control.After 2 days of cell culture,qRT-PCR and Western blot were used to detect the expression level of TIM-3 in cells,while MTT assay was used to detect the cell proliferation.The colony forming ability of cells was detected by plate cloning test,while cell cycle was detected with PI single staining method.Western blot was used to detect the levels of CyclinD1 and p27s protein in the cells;ELISA was used to detect the levels of VEGF and IL-10 secreted by cells.Data showed the expression levels of TIM-3 mRNA and protein in sh-TIM-3 group were reduced,the proliferation of SW620 cells decreased,cell colony forming ability also decreased,the percentage of G 0/G 1 decreased,the level of CyclinD1 protein was decreased,the level of p27 protein was increased,the secretion of VEGF and IL-10 were decreased in sh-TIM3 group,as compared with control(P<0.05).Taken together,TIM-3 knockdown can inhibit the growth of colorectal cancer cells and reduce the secretion of immunosuppressive factors.