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目的探讨沉默结肠癌RKO细胞鞘氨醇激酶1(SphK1)基因对顺铂(DDP)敏感性的影响及作用机制。方法构建靶向SphK1基因的慢病毒载体并感染RKO细胞后,实时定量PCR检测SphK1的mRNA水平,Western blot法检测SphK1蛋白水平。然后将RKO细胞分为SphK1低表达组(sh SphK1组)、阴性感染对照组(sh Control组)和空白对照组。培养24、48、72 h,MTT法检测细胞增殖活性;(0、2、4、8、16、32)μg/m L DDP处理细胞后,MTT法再次检测细胞增殖活性,TUNEL检测细胞凋亡,Western blot法检测KI-67抗原(ki67)、Bcl-2、胱天蛋白酶9(caspase-9)和caspase-3蛋白的水平。结果敲低SphK1基因能抑制RKO细胞的增殖,促进细胞凋亡。DDP呈时间和浓度依赖性抑制各组细胞的增殖,与对照组和sh Control组相比,敲低SphK1的细胞增殖能力显著降低;DDP呈剂量依赖性诱导细胞凋亡,且敲低SphK1的细胞凋亡率明显高于对照组和sh Control组。此外,敲低SphK1后,抑制ki67和Bcl-2的表达,增加caspase-9和caspase-3的表达,经DDP处理后,ki67和Bcl-2蛋白的表达水平明显下降,caspase-9和caspase-3蛋白的表达水平显著增加。结论敲低SphK1基因降低Bcl-2水平、增加caspase-9、caspase-3水平,抑制RKO结肠癌细胞增殖并促进其凋亡,并增强RKO细胞对DDP的敏感性。
Objective To investigate the effect of SphK1 gene on the sensitivity of cisplatin (DDP) in silencing colon cancer RKO cells and its mechanism. Methods The lentiviral vector targeting SphK1 gene was constructed and transfected into RKO cells. The mRNA level of SphK1 was detected by real - time PCR, and the level of SphK1 protein was detected by Western blot. RKO cells were then divided into SphK1 low expression group (sh SphK1 group), negative infection control group (sh Control group) and blank control group. The cells were cultured for 24, 48, and 72 h. Cell proliferation was detected by MTT assay. Cell proliferation was detected by MTT assay after treated with DDP (0, 2, 4, 8, 16, 32) μg / The levels of KI-67, Bcl-2, caspase-9 and caspase-3 were detected by Western blot. Results knockdown of SphK1 gene can inhibit the proliferation of RKO cells and promote apoptosis. DDP inhibited cell proliferation in a dose-dependent and time-dependent manner. Compared with control and sh Control groups, DDP knockdown SphK1 significantly reduced cell proliferation; DDP induced cell apoptosis in a dose-dependent manner, and cells knocked down SphK1 The apoptosis rate was significantly higher than the control group and sh Control group. In addition, knockdown of SphK1 inhibited the expression of ki67 and Bcl-2, and increased the expression of caspase-9 and caspase-3. After DDP treatment, the expression of ki67 and Bcl-2 protein were significantly decreased, while the expressions of caspase-9 and caspase- 3 protein expression levels increased significantly. Conclusion Knockdown of SphK1 gene can decrease the level of Bcl-2, increase the level of caspase-9 and caspase-3, inhibit the proliferation and promote the apoptosis of RKO colon cancer cells, and enhance the sensitivity of RKO cells to DDP.