摘要
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 sphingosine kinase 1 （SphK1） gene silence on the sensitivity to cisplatin （DDP） in RKO colon cancer cell line and the potential mechanism. Methods Targeted SphK1 gene lentivirus virus was constructed to infect RKO cells. The relative mRNA expression of SphK1 was detected by quantitative real-time PCR （qRT-PCR） and the protein level of SphK1 was determined by Western blotting. Then RKO cells were divided into three groups： down-regulated SphK1 group （shSphK] group）, negative control group （shControl group） and blank control group （control group）. Cells of these groups were incubated for 0, 24, 48 and 72 hours with 0, 2, 4, 8, 16, 32 μg/mL DDP. After treatment, cell viability was evaluated by MFI assay. Cell apoptosis index was determined by TUNEL. The expressions of ki67, Bcl-2, caspase-9 and caspase-3 were tested by Western blotting. Results Down-regulation of SphK1 inhibited cell proliferation and enhanced apoptosis of RKO cells, expecially after exposed to DDP. Silence of SphK1 sensitized RKO cells to DDP in a concentration- and time-dependent manner. Cell proliferation of shSphKl group was obviously reduced compared with control group or shControl group, and cell apoptosis rate of shSphK1 group significantly increased compared with control group or shControl group. Moreover, with the down-regulation of SphK1, the expressions of ki67 and Bcl-2 were depressed; the expressions of caspase-9 and caspase-3 were raised, especially after treated with DDP. Conclusion Down-regulation of SphK1 may decrease the expression of Bcl-2, increase the expressions of caspase-9 and caspase-3, inhibit cell proliferation, and promote cell apoptosis, thus improving chemosensitivity of colon cancer RKO cells to DDP.
作者
覃琳
刘诗权
覃蒙斌
伍文红
覃楠
符振华
徐春燕
黄杰安
赖铭裕
QIN Lin LIU Shiquan QIN Mengbin WU Wenhong QIN Nan FU Zhenhua XU Chunyan HUANG Jie＇an LAI Mingyu(Department of Gastroenterology, First Affiliated Hospital, Guangxi Medical University, Nanning 530021, China)
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2017年第5期623-629,共7页
Chinese Journal of Cellular and Molecular Immunology
基金
国家自然科学基金(81460380)
广西省自然科学基金(2011GXNSFA018182)
