饥饿条件下敲低SphK1通过调控自噬抑制结肠癌细胞的增殖

Knockdown of SphK1 inhibits the proliferation of colon cancer cells by regulating autophagy during starvation

目的:探讨在营养缺乏的肿瘤微环境中,敲低鞘氨醇激酶1(SphK1)后调控的自噬对人结肠癌RKO细胞增殖的影响及其机制。方法:Western blot检测正常结肠黏膜细胞ncm460和结肠癌RKO细胞SphK1表达、细胞外信号调节激酶(ERK)信号通路和自噬水平差异;用平衡盐溶液EBSS以不同干预时间(0 h、2 h、4 h、6 h)处理结肠癌RKO细胞,选取自噬激活最明显的时间进行后续实验;采用慢病毒siRNA-NC和siRNA-SphK1转染结肠癌RKO细胞;在EBSS干预4 h模拟营养缺乏的肿瘤微环境下,用CCK8和Western blot分别检测对照组(NC-RKO)和低表达SphK1组[SphK1(-)-RKO]细胞的存活率和SphK1、p-ERK和自噬水平的变化;再以ERK抑制剂u0126(10μmol/L)、自噬抑制剂3-MA(10 mmol/L)处理NC-RKO细胞,用CCK8和Western blot分别检测各组细胞的存活率和p-ERK、自噬水平的变化。结果:与正常结肠黏膜细胞ncm460相比,结肠癌细胞中SphK1、p-ERK、自噬水平均增加;在营养缺乏的肿瘤微环境下,低表达的SphK1抑制细胞的存活率,降低p-ERK、自噬水平;u0126和3MA处理后抑制NC-RKO细胞的增殖活性,降低自噬水平,u0126处理能够降低p-ERK的表达,而3MA处理后p-ERK无明显改变,差异均有统计学意义(P<0.05)。结论:在营养缺乏的肿瘤微环境中,抑制SphK1能有效抑制结肠癌细胞的增殖,其机制可能是通过调控ERK信号通路而抑制自噬来实现的。

Objective:To investigate the effect and the mechanism of autophagy regulated by knocking down sphingosine kinase 1(SphK1)on proliferation of human colon cancer RKO cell in the tumor microenvironment of nutritionally deficient.Methods:Western blot was used to detect the difference of Sph K1 expression,extracellular signal-regulated kinase(ERK)signaling pathway,and autophagy level between normal colon mucosa cells and colon cancer RKO cells.Colon cancer RKO cells were treated with balanced salt solution EBSS at different intervention time(0 h,2 h,4 h,6 h),and the most obvious time of autophagy activation was selected for subsequent experiments.Colon cancer RKO cells were transfected with lentiviral si RNA-NC and si RNA-Sph K1.In the tumor microenvironment simulated by EBSS intervention for 4 h,CCK8 and Western blot were used to detect the changes of cell survival rate,Sph K1,p-ERK,and autophagy level in the control group(NC-RKO)and low-expression Sph K1 group(Sph K1(-)-RKO).NC-RKO cells were treated with ERK inhibitor u0126(10μmol/L)and autophagy inhibitor 3-MA(10 mmol/L),then CCK8 and Western blot were used to detect the cell survival rate,p-ERK,and autophagy levels in each group.Results:Compared with normal colonic mucosal cells,the levels of Sph K1,p-ERK,and autophagy were increased in colon cancer cells.Under the nutritionally deficient tumor microenvironment,the low expression of Sph K1 inhibited cell survival,reduced p-ERK,and reduced autophagy levels.u0126 and 3 MA treatment could inhibit the proliferation of NC-RKO cells and reduce the level of autophagy.u0126 treatment could reduce the expression of p-ERK,while 3 MA treatment had no effect of p-ERK changed,and the differences were statistically significant(P<0.05).Conclusion:In a nutritionally deficient tumor microenvironment,inhibition of Sph K1 can effectively inhibit the proliferation of colon cancer cells,and the mechanism may be associated with regulating ERK signaling pathway to inhibiting autophagy.