SphK1对结肠癌lovo细胞的增殖、迁移和凋亡的影响

Sphingosine kinase 1 enhances cell proliferation and migration and suppresses apoptosis in human colon cancer cell line lovo

目的:探讨鞘胺醇激酶1(SphK1)对结肠癌lovo细胞增殖、迁移和凋亡的影响及其作用机制.方法:培养人结肠癌lovo细胞株,实验分3组:对照组、PMA组和DMS组.PMA组加入佛波醇-12-豆蔻酸酯-13-乙酸酯(PMA,100nmol/L),DMS组加入N,N-二甲基鞘胺醇(DMS,50μmol/L),对照组加入等量的培养基.采用MTT法和克隆形成实验检测细胞生长增殖的变化,流式细胞术检测细胞凋亡,Tranwell小室模型观察细胞迁移能力的变化,RT-PCR检测mRNA的表达,Westernblot检测蛋白的表达.结果:PMA显著促进细胞的增殖、迁移并抑制细胞的凋亡,DMS则显著抑制细胞的增殖、迁移并促进细胞的凋亡(对照组、PMA组和DMS组的细胞增殖活力:0.71±0.03vs1.05±0.05vs0.46±0.04;克隆形成率:1.32%±0.26%vs2.17%±0.17%vs0.73%±0.13%;凋亡率:16.25%vs9.15%vs32.58%;迁移细胞数:72.19±3.36vs98.46±6.25vs40.48±4.27;均P<0.05).PMA显著促进黏着斑激酶(FAK)的活性和表达,相反DMS则抑制FAK的活性和表达[对照组、PMA组和DMS组FAKmRNA的表达强度:0.151±0.008vs0.212±0.014vs0.114±0.021;蛋白:0.332±0.022vs0.374±0.029vs0.296±0.018;磷酸化FAK(p-FAKTyr397)蛋白:0.186±0.032vs0.234±0.017vs0.112±0.023;均P<0.05].结论:SphK1可促进lovo细胞的增殖和迁移能力并抑制细胞的凋亡,其机制可能是通过激活FAK通路而发挥作用.

AIM：To investigate the effect of sphingosine kinase 1（SphK1） on the proliferation,apoptosis and migration of colon cancer cells and to explore the molecular mechanisms involved.METHODS：Cultured lovo cells were divided into three groups：PMA group,DMS group and control group.Cells of the PMA group were treated with 100 nmol/L of phorbol 12-myristate 13-acetate（PMA）.The DMS group was treated with 50 μmol/L N,N-dimethylsphingosine（DMS）,while the control group was treated with equal volume of culture medium.After treatment,cell proliferation was determined by MTT assay and colony formation assay,and cell apoptosis was detected by ？ ow cytometry.Cell migration was assessed using Transwell chamber assays.RT-PCR and Western blot were used to evaluate the mRNA and protein expression of Sphk1 and FAK,respectively.RESULTS：PMA significantly enhanced cell proliferation and migration but suppressed cell apoptosis,whereas DMS suppressed cell proliferation and migration but enhanced cell apoptosis.Cell viability,colony formation rate,apoptosis rate and number of migrated cells for the control group,PMA group and DMS group were as follows：cell viability：0.71 ± 0.03,1.05 ± 0.05 and 0.46 ± 0.04;colony formation rate：1.32% ± 0.26%,2.17% ± 0.17% and 0.73% ± 0.13%;apoptosis rate：16.25%,9.15% and 32.58%;number of migrated cells：72.19 ± 3.36 vs 98.46 ± 6.25 vs 40.48 ± 4.27（all P 0.05 vs the control group）.PMA significantly up-regulated the expression and activity of focal adhesion kinase（FAK）,while DMS down-regulated the expression and activity of FAK（FAK mRNA：0.151 ± 0.008 vs 0.212 ± 0.014 vs 0.114 ± 0.021;FAK protein：0.332 ± 0.022 vs 0.374 ± 0.029 vs 0.296 ± 0.018;phosphor-FAK protein：0.186 ± 0.032 vs 0.234 ± 0.017 vs 0.112 ± 0.023;all P 0.05 vs the control group）.CONCLUSION：SphK1 enhances cell proliferation and migration and suppresses cell apoptosis in human colon cancer cell line lovo possibly by activating FAK.