1-磷酸神经酰胺转运蛋白对人口腔鳞状细胞癌HSC-3细胞生物学行为的影响

Effect of ceramide 1-phosphate transfer protein on biological behavior of human oral squamous cell carcinoma HSC-3 cells

目的:探讨1-磷酸神经酰胺转运蛋白(CPTP)对人口腔鳞状细胞癌HSC-3细胞生物学行为的影响,阐明其相关作用机制。方法:体外培养HSC-3细胞,分为对照组和实验组,分别采用pFlag-CMV4和pFlag-CPTP质粒进行转染,采用抗性筛选法构建稳定转染(稳转) CPTP的细胞。采用Western blotting法和免疫荧光法检测2组细胞中CPTP蛋白表达水平,采用克隆形成实验和CCK-8法检测各组细胞中克隆形成数和细胞增殖活性,细胞划痕实验检测2组细胞划痕愈合率,Transwell小室实验检测2组细胞中侵袭细胞数。小鼠随机分为对照组和实验组,分别注射pFlag-CMV4稳转HSC-3细胞和pFlag-CPTP稳转HSC-3细胞,制备小鼠皮下移植瘤模型,检测2组小鼠移植瘤体积和质量。采用生物信息学方法对头颈部鳞状细胞癌(HNSCC)组织中CPTP差异表达基因进行富集分析。采用实时荧光定量PCR (RT-qPCR)法检测2组细胞中p53、血小板反应蛋白1 (THBS1)和细胞周期蛋白G2 (CCNG2) mRNA表达水平。结果:与对照组比较,实验组细胞中CPTP蛋白表达量明显增加。与对照组比较,实验组细胞中克隆形成数明显增加(P<0.01),细胞增殖活性和划痕愈合率明显升高(P<0.05或P<0.01),侵袭细胞数明显增加(P<0.01)。注射肿瘤细胞2、3和4周,与对照组比较,实验组小鼠移植瘤体积明显增加(P<0.05或P<0.01);注射肿瘤细胞4周,与对照组比较,实验组小鼠移植瘤质量明显增加(P<0.05)。生物信息学分析,在HNSCC组织中CPTP的作用可能是通过p53等信号通路介导。与对照组比较,实验组细胞中p53、THBS1和CCNG2 mRNA表达水平明显降低(P<0.01)。结论:过表达CPTP能促进HSC-3细胞增殖、迁移、侵袭和成瘤能力,CPTP通过抑制p53信号通路促进口腔鳞状细胞癌HSC-3细胞生长。

Objective:To discuss the effect of human ceramide 1-phosphate transfer protein(CPTP)on the biological behavior of oral squamous cell carcinoma HSC-3 cells,and to clarify its related mechanism.Methods:The HSC-3 cells were cultured in vitro and divided into control group and experiment group,and the cells in two groups were transfected with pFlag-CMV4 and pFlag-CPTP plasmids,respectively;the cells stably transfected with CPTP were constructed by using resistance screening method.Western blotting and immunofluorescence methods were used to detect the expression levels of CPTP protein in cells in two groups;clone formation experiment and CCK-8 assay were used to detect the numbers of clone formation and proliferation activities of cells in two groups;cell scratch experiment was used to detect the scratch healing rates of cells in two groups;Transwell chamber experiment was used to detect the numbers of invasion cells in two groups.The mice were randomly divided into control group and experiment group,and the mice were injected with the HSC-3 cells stably transfected with pFlag-CMV4 and the HSC-3 cells stably transfected with pFlag-CPTP respectively to construct the subcutaneous transplanted tumor models in the mice.The volumes and weights of transplant tumors of the mice in two groups were detected.The enrichment analysis on CPTP differentially expressed genes in head and neck squamous cell carcinoma(HNSCC)cells was analyzed by using bioinformatics method;real-time fluorescence quantitative PCR(RT-qPCR)method was used to detect the expression levels of p53,thrombospondin-1(THBS1),and cyclin G2(CCNG2)mRNA in cells in two groups.Results:Compared with control group,the expression amount of CPTP protein in the cells in experiment group was increased.Compared with control group,the number of clone formation in the cells in experiment group was significantly increased(P<0.01),the proliferation activity and scratch healing rate of the cells were significantly increased(P<0.05 or P<0.01),and the number of invasion cells was increased(P<0.01).After injecting tumor cells for 2,3,and 4 weeks,compared with control group,the volumes of the transplanted tumor of the mice in experiment group were increased(P·<0.05 or P<0.01);after injecting tumor cells for 4 weeks,compared with control group,the weight of transplanted tumor of the mice in experiment group was increased(P·<0.05).The bioinformatics analysis results showed that the role of CPTP in the HNSCC tissue may be mediated through the signaling pathways such as p53.Compared with control group,the expression levels of p53,THBS1,and CCNG2 mRNA in the cells in experiment group were significantly decreased(P<0.01).Conclusion:Over-expression of CPTP can promote the proliferation,migration,invasion,and tumorigenesis of the HSC-3 cells.CPTP promotes the growth of the oral squamous cell carcinoma HSC-3 cells by inhibiting the p53 signaling pathway.