口腔疾病防治 ›› 2018, Vol. 26 ›› Issue (1): 31–37.doi: 10.12016/j.issn.2096-1456.2018.01.006

• 基础研究 • 上一篇    下一篇

TPX2在舌鳞状细胞癌组织中的表达及其对Cal27细胞增殖和凋亡的影响

尹颂豪1(), 郭伟洪2   

  1. 1. 东莞市人民医院口腔颌面外科,广东 东莞(523000)
    2. 南方医科大学第一临床医学院,广东 广州(510515)
  • 收稿日期:2017-05-16 修回日期:2017-07-18 发布日期:2018-08-31
  • 作者简介:

    【作者简介】 黎景景,医师,硕士, Email:492207014@qq.com

  • 基金资助:
    2017年广东省大学生科技创新培育专项资金(pdjh2017a0096)

Investigation of the expression of TPX2 in tongue squamous cell carcinoma and its effect on Cal27 cell proliferation and apoptosis

Songhao YIN1(), Weihong GUO2   

  1. 1. Department of Oral and Maxillofacial Surgery, People's Hospital, Dongguan 523000, China;
    2. Department of First Medical College, Southern Medical University, Guangzhou 510515, China
  • Received:2017-05-16 Revised:2017-07-18 Published:2018-08-31

摘要:

目的 研究Xklp2靶蛋白(targeting protein for xenopus kinesin-like protein2,TPX2)在舌鳞状细胞癌组织中的表达及意义,并探讨其在舌鳞癌细胞株Cal27增殖和凋亡过程中的作用。方法 收集新鲜的舌鳞状细胞癌组织及其配对癌旁组织30例于液氮中保存,采用荧光定量PCR、免疫印迹western blot方法检测TPX2在舌鳞状细胞癌癌组织和癌旁组织中mRNA、蛋白水平的表达情况,分析TPX2表达水平与临床病理参数的相关性;进一步用小干扰RNA(small interfering RNA,siRNA)敲低舌鳞状细胞癌细胞株Cal27细胞TPX2的表达量,以MTT法、流式细胞术和Western blot法分别检测细胞增殖、凋亡以及凋亡相关蛋白(cleaved caspase 3、caspase 3)表达水平的变化。结果 在转录水平,TPX2在30例舌鳞状细胞癌患者癌组织中呈高表达状态,23例患者的癌组织TPX2的表达水平高于其对应的癌旁组织(t=3.254,P=0.002 9);在蛋白水平,TPX2在30例舌鳞状细胞癌患者癌组织中呈高表达状态,20例患者的癌组织TPX2的表达水平高于其对应的癌旁组织(t=2.862,P=0.007 7),且TPX2的高表达与舌鳞状细胞癌的T分期、淋巴结转移具有相关性;转染TPX2 siRNA 48 h可以抑制Cal27细胞增殖能力,增加凋亡细胞比例(F=342.9,P < 0.000 1),同时上调Cleaved caspase 3(F=46.98,P=0.001 4)、Caspase 3(F=33.35,P=0.002 7)相关凋亡蛋白的表达。结论 TPX2在舌鳞状细胞癌组织中呈高表达,干扰Cal27细胞中TPX2的表达可以抑制细胞增殖,诱导细胞凋亡,提示TPX2可能是舌鳞状细胞癌基因治疗的新靶点之一。

关键词: 舌鳞状细胞癌, TPX2, 细胞增殖, 凋亡, Cal27细胞

Abstract:

Objective To investigate the expression of targeting protein for xenopus kinesin-like protein2 (TPX2) in tongue squamous cell carcinoma (TSCC) tissues and explore its effect on cell proliferation and apoptosis. Methods 30 cases of TSCC tissues, paired normal tissues were collected in Dongguan People's Hospital during 2013-2016. The mRNA and protein expression level of TPX2 was determined by qRT-PCR and western blot, respectively and analyzed The correlation of TPX2 expression level and clinic opathological parameters. Cal27 cell was transfected with siRNAs to knockdown the expression of TPX2, then cell proliferation, cell apoptosis and related proteins (cleaved caspase 3 and caspase 3) were detected by MTT assay, flow cytometry and western blot. Results TPX2 was highly expressed in (t=3.254, P=0.002 9) tumor tissues at mRNA level compared to adjacent normal parts. In protein level, TPX2 was highly expressed (66.7%) in tumor tissues, TPX2 expression level of 20 case was higher than the corresponding tissue adjacent to carcinoma (20/30, t=2.862, P=2.862), and high expression of TPX2 was related to T staging, lymph node metastasis of tongue cancer. Knockdown TPX2 effectively reduced cell proliferation, increased apoptosis rate (F=342.9, P < 0.000 1) and upregulated the expression of apoptosis-related proteins cleaved caspase 3 (F=46.98, P=0.001 4) and caspase 3 (F=33.35, P=0.002 7). Conclusion Overexpression of TPX2 was found in TSCC tissues. Silencing of TPX2 might inhibit cell proliferation and promote cell apoptosis. TPX2 could be a new target for gene therapy of TSCC.

Key words: TSCC, TPX2, Cell proliferation, Apoptosis, Cal27 cell

中图分类号: 

  • R739.8

图1

TPX2在舌鳞状细胞癌组织和癌旁组织中的基因、蛋白表达情况 a:TPX2在舌鳞状细胞癌和癌旁组织中的mRNA表达情况;b:TPX2在舌鳞状细胞癌和癌旁组织中的蛋白表达情况。TPX2:Xklp2靶蛋白。"

图2

TPX2 siRNA对TPX2 mRNA和蛋白质表达水平的影响 a:转染48 h后,Cal27细胞TPX2 mRNA表达水平; b:转染72 h,Cal27细胞TPX2蛋白质表达水平。*与NC组比较, P < 0.05。TPX2: Xklp2靶蛋白。"

图3

TPX2对Cal27细胞增殖能力的影响#与NC组比较,P < 0.001;TPX2:Xklp2靶蛋白。"

图4

转染TPX2 siRNA 48 h后TPX2对Cal27细胞凋亡比例的影响 Q1:死亡细胞;Q2:晚期凋亡细胞; Q3:早期凋亡细胞; Q4:活细胞。*与NC组比较, P < 0.05。TPX2: Xklp2靶蛋白。"

表1

TPX2表达与舌鳞状细胞癌患者临床病理参数的关系"

病理参数 n TPX2mRNA高表达 χ2 P TPX2蛋白高表达 χ2 P
性别 18 14 0.031 0.860 12 0.000 1.000
12 9 8
年龄 <60岁 14 10 0.403 0.526 10 0.268 0.605
≥60岁 16 13 10
组织分化 高中分化 22 17 0.017 0.896 13 2.131 0.144
低分化 8 6 7
T分期 T1,T2 13 7 6.679 0.010 6 4.344 0.037
T3,T4 17 16 14
淋巴结转移 16 14 5.000 0.025 15 11.317 0.001
14 7 5

图5

转染72 h后TPX2对Cal27细胞内凋亡相关蛋白的影响*与NC组比较, P < 0.05。TPX2:Xklp2靶蛋白。"

[1] Siegel RL, Miller KD, Jemal A.Cancer statistics, 2017[J]. CA Cancer J Clin, 2017, 67(1): 7-30.
[2] Jemal A, Bray F, Center MM, et al.Global cancer statistics[J]. Cancer J Clin, 2011, 61(2): 69-90.
[3] Tertipis N, Hammar U, Nasman A, et al.A model for predicting clinical outcome in patients with human papillomavirus-positive tonsillar and base of tongue cancer[J]. Eur J Cancer, 2015, 51(12): 1580-1587.
[4] Upadhyay P, Nair S, Kaur E, et al.Notch pathway activation is essential for maintenance of stem-like cells in early tongue cancer[J]. Oncotarget, 2016, 7(31): 50437-50449.
[5] Yu X, Li Z.MicroRNA expression and its implications for diagnosis and therapy of tongue squamous cell carcinoma[J]. J Cell Mol Med, 2016, 20(1): 10-16.
[6] Fan S, Chen WX, Lv XB, et al.miR-483-5p determines mitochondrial fission and cisplatin sensitivity in tongue squamous cell carcinoma by targeting FIS1[J]. Cancer Lett, 2015, 362(2): 183-191.
[7] Neumayer G, Belzil C, Gruss OJ, et al.TPX2: of spindle assembly, DNA damage response, and cancer[J]. Cell Mol Life Sci, 2014, 71(16): 3027-3047.
[8] Liang B, Zheng WJ, Fang L, et al.Overexpressed targeting protein for Xklp2 (TPX2) serves as a promising prognostic marker and therapeutic target for gastric cancer[J]. Cancer Biol Ther, 2016, 17(8): 824-832.
[9] Chang HP, Wang JZ, Tian Y, et al.The TPX2 gene is a promising diagnostic and therapeutic target for cervical cancer[J]. Oncol Rep, 2012, 27(5): 1353-1359.
[10] Liang B, Zheng WJ, Fang L, et al.Overexpressed targeting protein for Xklp2 (TPX2) serves as a promising prognostic marker and therapeutic target for gastric cancer[J]. Cancer Biol Ther, 2016, 17(8): 824-832.
[11] Yan L, Li SL, Xu CB, et al.Target protein for Xklp2 (TPX2), a microtubule-related protein, contributes to malignant phenotype in bladder carcinoma[J]. Tumor Biology, 2013, 34(6): 4089-4100.
[12] Zhang P, Zhang L, Liu H, et al.Clinicopathologic characteristics and prognosis of tongue squamous cell carcinoma in patients with and without a history of radiation for nasopharyngeal carcinoma: a matched Case-Control study[J]. Cancer Res Treat, 2017, 49(3): 695-705.
[13] Yu JJ, Liu Y, Gong ZJ, et al.Overexpression long non-coding RNA LINC00673 is associated with poor prognosis and promotes invasion and metastasis in tongue squamous cell carcinoma[J]. Oncotarget, 2017, 8(10): 16621-16632.
[14] Hsu PK, Chen HY, Yeh YC, et al.TPX2 expression is associated with cell proliferation and patient outcome in esophageal squamous cell carcinoma[J]. J Gastroenterol, 2014, 49(8): 1231-1240.
[15] Wieczorek M, Bechstedt S, Chaaban S, et al.Microtubule-associated proteins control the kinetics of microtubule nucleation[J]. Nat Cell Biol, 2015, 17(7): 907-916.
[16] 林冬梅, 马莹, 肖汀, 等. TPX2在肺鳞状细胞癌及其癌前病变中的表达和意义[J]. 中华病理学杂志, 2006, (9): 540-544.
[17] Warner SL, Stephens BJ, Nwokenkwo SA, et al.Validation of TPX2 as a potential therapeutic target in pancreatic cancer cells[J]. Clin Cancer Res, 2009, 15(21): 6519-6528.
[18] Wei P, Zhang N, Xu Y, et al.TPX2 is a novel prognostic marker for the growth and metastasis of colon cancer[J]. J Transl Med, 2013, 11: 313.
[19] Liu QQ, Tu KS, Zhang HY, et al.TPX2 as a novel prognostic biomarker for hepatocellular carcinoma[J]. Hepatology Research, 2015, 45(8): 906-918.
[20] Pascreau G, Eckerdt F, Lewellyn AL, et al.Phosphorylation of p53 is regulated by TPX2-Aurora A in xenopus oocytes[J]. J Biol Chem, 2009, 284(9): 5497-5505.
[21] Neumayer G, Helfricht A, Shim SY, et al.Targeting protein for xenopus kinesin-like protein 2 (TPX2) regulates gamma-Histone 2AX (gamma-H2AX) levels upon ionizing radiation[J]. J Biol Chem, 2012, 287(50): 42206-42222.
[22] Wong RS.Apoptosis in cancer: from pathogenesis to treatment[J]. J Exp Clin Cancer Res, 2011, 30(1): 1-14.
[23] Fulda S, Debatin KM.Targeting apoptosis pathways in cancer therapy[J]. Cancer J Clin, 2005, 4(3): 569-576.
[24] Yang Q, Wang Y, Yang Q, et al.Cuprous oxide nanoparticles trigger ER stress-induced apoptosis by regulating copper trafficking and overcoming resistance to sunitinib therapy in renal cancer[J]. Biomaterials, 2017, 146: 72-85.
[25] Park M, Yoon HJ, Kang MC, et al.MiR-338-5p enhances the radiosensitivity of esophageal squamous cell carcinoma by inducing apoptosis through targeting surviving[J]. Sci Rep, 2017, 7(1): 10932.
[1] 王晓瑾, 游昕超, 陈凯, 黄坤宋, 潘宣. 木犀草素对人舌鳞癌细胞侵袭和迁移的影响[J]. 口腔疾病防治, 2018, 26(7): 434-439.
[2] 周丽娟(综述), 谢思明(审校). 间隙连接蛋白43在口腔鳞状细胞癌中的研究进展[J]. 口腔疾病防治, 2018, 26(5): 338-340.
[3] 王亚楠, 刘浩, 张军. 自噬相关蛋白PTEN、MAP1LC3 在舌癌中的表达与意义[J]. 口腔疾病防治, 2017, 25(5): 294-299.
[4] 王焕玲, 张建平. 槟榔碱对人牙周膜成纤维细胞凋亡的影响[J]. 口腔疾病防治, 2017, 25(4): 223-226.
[5] 文军, 徐帅妹, 刘影. Wnt通路抑制剂XAV-939对牙髓干细胞成脂分化的影响[J]. 口腔疾病防治, 2016, 24(8): 459-463.
[6] 韩倩倩, 刘钊, 江丽, 汤慧怡, 李晓娜. LMK-235 对牙周膜细胞成骨和成牙本质分化的影响[J]. 口腔疾病防治, 2016, 24(7): 390-394.
[7] 于丽娜, 张文轩, 于洋, 秦文光, 郭雪琪. shRNA沉默LASP-1对舌鳞状细胞癌SCC9细胞增殖和迁移的影响[J]. 口腔疾病防治, 2016, 24(6): 336-340.
[8] 朴正国, 邹瑞, 闫璟, Seok-Kee Baik, Hye-Jin Tak, Sang-Hwy Lee. O-GlcNAc通过抑制Wnt信号通路影响上唇发育的实验研究[J]. 口腔疾病防治, 2016, 24(5): 273-279.
[9] 张志利, 卢文辉, 曾威, 邓璋, 周斌, 王友元. Ezrin 对人舌鳞癌顺铂耐药细胞上皮—间质转化的影响[J]. 口腔疾病防治, 2016, 24(12): 688-694.
[10] 赵树蕃, 崔颖秋. 黄芩苷对氧化应激诱导牙龈上皮细胞凋亡的保护作用研究[J]. 口腔疾病防治, 2016, 24(12): 701-705.
Viewed
Full text


Abstract

Cited

  Shared   
  Discussed   
[1] 郭磊, 杨倞, 王远勤. 上颌后牙区数字化导板引导下倾斜种植精确性研究[J]. 口腔疾病防治, 2017, 25(7): 435 -438 .
[2] 张秀丽, 徐小川, 姚源, 李昀生. 北京东城区高三学生牙周健康状况调查[J]. 口腔疾病防治, 2017, 25(8): 537 -540 .
[3] 孙立众, 王琦, 王若帆(综述), 米方林(审校). 次氯酸钠去蛋白化对正畸托槽粘接影响的研究进展[J]. 口腔疾病防治, 2018, 26(6): 396 -400 .