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

doi:10.13481/j.1671-587X.20230407

摘要/Abstract

摘要：

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。

关键词: 口腔鳞状细胞癌, 1-磷酸神经酰胺转运蛋白, 细胞侵袭, 信号通路

Abstract:

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.

Key words: Oral squamous cell carcinoma, Ceramide-1-phosphate transfer protein, Cell invasion, Signaling pathway

中图分类号:

R734.2

张湘豫,胡溢洪,韩语诚,邹先琼. 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[J]. 吉林大学学报(医学版), 2023, 49(4): 875-883.

Xiangyu ZHANG,Yihong HU,Yucheng HAN,Xianqiong ZOU. Effect of ceramide 1-phosphate transfer protein on biological behavior of human oral squamous cell carcinoma HSC-3 cells[J]. Journal of Jilin University(Medicine Edition), 2023, 49(4): 875-883.

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参考文献 28

1	ZHANG Y Q， ZHANG X Y， LU M Y， et al. Ceramide-1-phosphate and its transfer proteins in eukaryotes［J］. Chem Phys Lipids， 2021， 240： 105135.
2	MISHRA S K， GAO Y G， DENG Y B， et al. CPTP： a sphingolipid transfer protein that regulates autophagy and inflammasome activation［J］. Autophagy， 2018， 14（5）： 862-879.
3	SIMANSHU D K， KAMLEKAR R K， WIJESINGHE D S， et al. Non-vesicular trafficking by a ceramide-1-phosphate transfer protein regulates eicosanoids［J］. Nature， 2013， 500（7463）： 463-467.
4	GOMEZ-LARRAURI A， PRESA N， DOMINGUEZ-HERRERA A， et al. Role of bioactive sphingolipids in physiology and pathology［J］. Essays Biochem， 2020， 64（3）： 579-589.
5	MACEYKA M， SPIEGEL S. Sphingolipid metabolites in inflammatory disease［J］. Nature， 2014， 510（7503）： 58-67.
6	WANG J， LI C， JIANG Y J， et al. Effect of ceramide-1-phosphate transfer protein on intestinal bacterial translocation in severe acute pancreatitis［J］. Clin Res Hepatol Gastroenterol， 2017， 41（1）： 86-92.
7	ZHANG Y Q， JI S Y， ZHANG X Y， et al. Human CPTP promotes growth and metastasis via sphingolipid metabolite ceramide and PI4KA/AKT signaling in pancreatic cancer cells［J］. Int J Biol Sci， 2022， 18（13）： 4963-4983.
8	VASAIKAR S V， STRAUB P， WANG J， et al. LinkedOmics： analyzing multi-omics data within and across 32 cancer types［J］. Nucleic Acids Res， 2018， 46（D1）： D956-D963.
9	CHUN K H. Discovery of cellular RhoA functions by the integrated application of gene set enrichment analysis［J］. Biomol Ther， 2022， 30（1）： 98-116.
10	ZHAO L Z， LIANG J S， ZHONG W， et al. Expression and prognostic analysis of BGN in head and neck squamous cell carcinoma［J］. Gene， 2022， 827： 146461.
11	AUPÉRIN A. Epidemiology of head and neck cancers： an update［J］. Curr Opin Oncol， 2020， 32（3）： 178-186.
12	WARNAKULASURIYA S. Living with oral cancer： epidemiology with particular reference to prevalence and life-style changes that influence survival［J］. Oral Oncol， 2010， 46（6）： 407-410.
13	BAUMEISTER P， ZHOU J F， CANIS M， et al. Epithelial-to-mesenchymal transition-derived heterogeneity in head and neck squamous cell carcinomas［J］. Cancers， 2021， 13（21）： 5355.
14	ZHANG S Z， XIE L， SHANG Z J. Burden of oral cancer on the 10 most populous countries from 1990 to 2019： estimates from the global burden of disease study 2019［J］.Int J Environ Res Public Health，2022，19（2）： 875.
15	BRAY F， FERLAY J， SOERJOMATARAM I， et al. Global cancer statistics 2018： GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries［J］. CA Cancer J Clin， 2018， 68（6）： 394-424.
16	ASTHANA S， LABANI S， KAILASH U， et al. Association of smokeless tobacco use and oral cancer： a systematic global review and meta-analysis［J］. Nicotine Tob Res， 2019， 21（9）： 1162-1171.
17	SAXENA R， PRASOODANAN P K V， GUPTA S V， et al. Assessing the effect of smokeless tobacco consumption on oral microbiome in healthy and oral cancer patients［J］. Front Cell Infect Microbiol， 2022， 12： 841465.
18	WEBER M， LUTZ R， OLMOS M， et al. Beyond PD-L1-identification of further potential therapeutic targets in oral cancer［J］. Cancers， 2022， 14（7）： 1812.
19	LIU S， SHI C Y， HOU X R， et al. Transcriptional and H3K27ac related genome profiles in oral squamous cell carcinoma cells treated with metformin［J］. J Cancer， 2022， 13（6）： 1859-1870.
20	BRENNAN P A， DYLGJERI F， COLETTA R D，et al. Surgical tumour margins and their significance in oral squamous cell carcinoma［J］. J Oral Pathol Med， 2022， 51（4）： 311-314.
21	STARZYŃSKA A， SOBOCKI B K， ALTERIO D. Current challenges in head and neck cancer management［J］. Cancers， 2022， 14（2）： 358.
22	FAN Z L， YANG L Y， ZHANG N， et al. NEFL promotes invasion and migration of esophageal squamous carcinoma cells via the EGFR/AKT/S6 pathway［J］. Hereditas， 2022， 44（4）： 322-334.
23	QIU H Q， ZHANG L， WANG D Z， et al. ZNF488 promotes the invasion and migration of pancreatic carcinoma cells through the akt/mTOR pathway［J］. Comput Math Methods Med， 2022， 2022： 4622877.
24	ZHANG Y H， MOHIBI S， VASILATIS D M， et al. Ferredoxin reductase and p53 are necessary for lipid homeostasis and tumor suppression through the ABCA1-SREBP pathway［J］. Oncogene， 2022， 41（12）： 1718-1726.
25	WANG M X， ATTARDI L D. A balancing act： p53 activity from tumor suppression to pathology and therapeutic implications［J］. Annu Rev Pathol， 2022， 17： 205-226.
26	李震，刘江凯. p53信号通路调控铁死亡在肝细胞癌中的作用机制［J］.临床肝胆病杂志，2023，39（4）： 956-960.
27	WANG W， XIAO L N， PAN D， et al. ASF1B enhances migration and invasion of lung cancers cell via regulating the P53-mediated epithelial-mesenchymal transformation （EMT） signaling pathway［J］. Neoplasma， 2022， 69（2）： 361-369.
28	MARTINEZ J D， MO Q X， XU Y X， et al. Common genomic aberrations in mouse and human breast cancers with concurrent P53 deficiency and activated PTEN-PI3K-AKT pathway［J］. Int J Biol Sci， 2022， 18（1）： 229-241.

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