CMTM4在口腔鳞状细胞癌发生发展中的作用及机制研究

doi:10.13591/j.cnki.kqyx.2023.06.001

摘要/Abstract

摘要：

目的探讨CMTM4对口腔鳞状细胞癌(oral squamous cell carcinoma, OSCC)的影响。方法采用免疫组织化学法分析CMTM4在OSCC组织中的表达情况,卡方检验及费希尔精确概率检验分析CMTM4表达差异与患者各临床病理参数的关系。在OSCC细胞系HN6和CAL27中分别转染CMTM4过表达质粒及敲低质粒,观察CMTM4过表达和敲低时对细胞表型行为的影响。通过CCK8实验、平板克隆形成实验、划痕实验及Transwell侵袭实验分析HN6及CAL27细胞生物学行为的变化。通过蛋白免疫印迹实验检测CMTM4改变后细胞周期相关蛋白及AKT通路蛋白的变化情况。裸鼠皮下成瘤实验进一步验证CMTM4对OSCC体内成瘤的作用。结果与癌旁组织相比,CMTM4在OSCC组织中高表达,且CMTM4表达差异与患者的年龄、临床分期及病理分级显著相关(P<0.001)。在HN6和CAL27细胞中,与对照组相比,CMTM4过表达后细胞的增殖速度变快,迁移和侵袭能力更强。相对应地,当CMTM4敲低后,与对照组相比,HN6和CAL27细胞的增殖、迁移、侵袭能力均下降。过表达CMTM4后细胞周期相关蛋白CDK4、CDK6和Cyclin D1以及AKT通路相关蛋白pAKT表达也有所升高。CMTM4敲低后这些蛋白表达水平明显降低。动物实验结果显示,与对照组相比,CMTM4过表达后OSCC细胞体内成瘤体积增大,CMTM4敲低后成瘤体积明显减小。结论 CMTM4在OSCC组织中高表达,可以促进OSCC细胞的增殖、迁移及侵袭能力,可能通过调控细胞周期及AKT通路发挥作用。

关键词: CMTM4, 口腔鳞状细胞癌, 细胞周期蛋白, AKT通路, 分子靶向治疗

Abstract:

Objective To investigate the effects of CMTM4 on oral squamous cell carcinoma(OSCC). Methods The expression of CMTM4 in OSCC was analyzed by immunohistochemistry, and the relationship between the difference of CMTM4 expression and clinicopathological parameters was analyzed by Chi-square testand Fisher's precision probability test. CMTM4 overexpression plasmid and small interference RNA plasmid were transfected into OSCC cell lines HN6 and CAL27respectively, and the effects of CMTM4 overexpression and knock-down expression on cell phenotypic behavior were observed. The changes in biological behavior of HN6 and CAL27 were analyzed by CCK8 test,plate clone formation test, wound healing assay and Transwell invasion test. Western blot was used to detect changes of related cyclins and AKT pathway proteins after the changes of CMTM4. The subcutaneous tumorigenesis experiment of nude mice further verified the effect of CMTM4 on the tumorigenesis of OSCC in vivo. Results Compared with para-carcinoma tissues, CMTM4 was highly expressed in OSCC, and the difference of CMTM4 expression was significantly correlated with age, clinical stage and pathological grade of the patients (P<0.001). In HN6 and CAL27, compared with the control group, the proliferation rate of cells became faster and the ability of migration and invasion was stronger after overexpression of CMTM4. When CMTM4 was knocked down, the cell proliferation, migration and invasion ability of HN6 and CAL27 cells decreased. Overexpression of CMTM4 increased the expression of cell cycle-related proteins CDK4, CDK6 and Cyclin D1 as well as pAKT, a protein related to AKT pathway. The expression level of these proteins decreased significantly after CMTM4's knock-down. The results of animal experiment showed that compared with the control group, the tumor volume of OSCC cells increased after CMTM4 overexpression, and decreased when CMTM4 was knocked down. Conclusion CMTM4 is highly expressed in OSCC and can promote the proliferation, invasion and migration of OSCC cell line, which may play a role by regulating cell cycle and AKT pathway.

Key words: CMTM4, oral squamous cell carcinoma, Cyclin, AKT pathway, molecular targeted therapy

中图分类号:

R739.85

胡海艳, 高腾, 朱载瓯, 丁旭, 吴煜农, 宋晓萌. CMTM4在口腔鳞状细胞癌发生发展中的作用及机制研究[J]. 口腔医学, 2023, 43(6): 481-487.

HU Haiyan, GAO Teng, ZHU Zai'ou, DING Xu, WU Yunong, SONG Xiaomeng. Study on the role of CMTM4 in oral squamous cell carcinoma and its mechanism[J]. Stomatology, 2023, 43(6): 481-487.

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参考文献 19

[1]	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 A Cancer J Clin, 2018, 68(6):394-424. doi: 10.3322/caac.v68.6
[2]	Uz U, Eskiizmir G. Association between interleukin-6 and head and neck squamous cell carcinoma:A systematic review[J]. Clin Exp Otorhinolaryngol, 2021, 14(1):50-60. doi: 10.21053/ceo.2019.00906
[3]	Chai AWY, Lim KP, Cheong SC. Translational genomics and recent advances in oral squamous cell carcinoma[J]. Semin Cancer Biol, 2020, 61:71-83. doi: S1044-579X(19)30260-3 pmid: 31542510
[4]	Lindemann A, Takahashi H, Patel AA, et al. Targeting the DNA damage response in OSCC with TP53 mutations[J]. J Dent Res, 2018, 97(6):635-644. doi: 10.1177/0022034518759068 pmid: 29489434
[5]	Han WL, Ding PG, Xu MX, et al. Identification of eight genes encoding chemokine-like factor superfamily members 1-8 (CKLFSF1-8) by in silico cloning and experimental validation[J]. Genomics, 2003, 81(6):609-617. doi: 10.1016/s0888-7543(03)00095-8 pmid: 12782130
[6]	Kittler R, Putz G, Pelletier L, et al. An endoribonuclease-prepared siRNA screen in human cells identifies genes essential for cell division[J]. Nature, 2004, 432(7020):1036-1040. doi: 10.1038/nature03159
[7]	Plate M, Li T, Wang Y, et al. Identification and characterization of CMTM4, a novel gene with inhibitory effects on HeLa cell growth through Inducing G2/M phase accumulation[J]. Mol Cells, 2010, 29(4):355-361. doi: 10.1007/s10059-010-0038-7 pmid: 20213316
[8]	Li T, Cheng YY, Wang PZ, et al. CMTM4 is frequently downregulated and functions as a tumour suppressor in clear cell renal cell carcinoma[J]. J ExpClin Cancer Res, 2015, 34:122.
[9]	Bei CH, Zhang Y, Wei RM, et al. Clinical significance of CMTM4 expression in hepatocellular carcinoma[J]. Onco Targets Ther, 2017, 10:5439-5443. doi: 10.2147/OTT
[10]	Li H, Liu YT, Chen L, et al. CMTM4 regulates epithelial-mesenchymal transition and PD-L1 expression in head and neck squamous cell carcinoma[J]. Mol Carcinog, 2021, 60(8):556-566. doi: 10.1002/mc.v60.8
[11]	Mezzadra R, Sun C, Jae LT, et al. Identification of CMTM6 and CMTM4 as PD-L1 protein regulators[J]. Nature, 2017, 549(7670):106-110. doi: 10.1038/nature23669
[12]	Tan SK, Guo XF, Bei CH, et al. Prognostic significance and immune characteristics of CMTM4 in hepatocellular carcinoma[J]. BMC Cancer, 2022, 22(1):905. doi: 10.1186/s12885-022-09999-y pmid: 35986302
[13]	Li MZ, Guo HH, Wang Q, et al. Pancreatic stellate cells derived exosomal miR-5703 promotes pancreatic cancer by downregulating CMTM4 and activating PI3K/Akt pathway[J]. Cancer Lett, 2020, 490:20-30. doi: S0304-3835(20)30339-6 pmid: 32585413
[14]	潘剑锋, 尚方正, 马荣, 等. CDK、CKI与周期蛋白在细胞周期进程中的调控机制研究进展[J/OL]. 生物工程学报:1-31[2023-03-06].https://doi.org/10.13345/j.cjb.220478.
[15]	Min MW, Rong Y, Tian CZ, et al. Temporal integration of mitogen history in mother cells controls proliferation of daughter cells[J]. Science, 2020, 368(6496):1261-1265. doi: 10.1126/science.aay8241 pmid: 32241885
[16]	Zhang JF, Bu X, Wang HZ, et al. Cyclin D-CDK4 kinase destabilizes PD-L1 via cullin 3-SPOP to control cancer immune surveillance[J]. Nature, 2018, 553(7686):91-95. doi: 10.1038/nature25015
[17]	Maiani E, Milletti G, Nazio F, et al. AMBRA1 regulates Cyclin D to guard S-phase entry and genomic integrity[J]. Nature, 2021, 592(7856):799-803. doi: 10.1038/s41586-021-03422-5
[18]	Zhang L, Lin SF, Zhang ZY, et al. The role of p21-activated kinase 4 in the progression of oral squamous cell carcinoma by targeting PI3K-AKT signaling pathway[J]. Clin Transl Oncol, 2023, 25(3):739-747. doi: 10.1007/s12094-022-02980-y pmid: 36593383
[19]	Zhang SN, Dong YQ, Zhao SY, et al. CXCL1 promoted the migration and invasion abilities of oral cancer cells and might serve as a promising marker of prognosis in tongue cancer[J/OL]. J Oral Pathol Med, 2023[2023-03-06]. https://onlinelibrary.wiley.com/doi/epdf/10.1111/jop.13418