下调体外培养头颈部鳞状细胞癌细胞iASPP的表达能抑制细胞增殖、侵袭,增加对紫杉醇的化学敏感性

doi:10.24920/003558

摘要/Abstract

摘要：

目的我们前期的研究表明:iASPP在人头颈鳞状细胞癌（HNSCC）中表达升高,且iASPP过表达与HNSCC的恶性生物学行为及不良预后密切相关。本研究旨在探讨下调iASPP表达对体外培养的HNSCC细胞系Tu686增殖和侵袭的影响。

方法采用慢病毒介导的特异性iASPP shRNA和control shRNA分别转染Tu686细胞,转染后的细胞分别作为shRNA-iASPP组和shRNA-NC组;未转染的Tu686细胞为CON组。采用CCK-8法、流式细胞术、Transwell侵袭实验检测下调iASPP基因表达对Tu686细胞的影响。

结果 CCK-8结果显示:shRNA-iASPP组细胞在转染后72 h（F=32.459,P=0.000）、96 h（F=51.407, P=0.000）、120 h（F=35.125,P=0.000）的增殖明显低于shRNA-NC组和CON组。流式细胞术结果显示:shRNA-iASPP组细胞凋亡率为9.42%±0.39%（F=299.490,P=0.000）,明显高于CON组（2.80%±0.42%）和shRNA-NC组（3.18%±0.28%）。shRNA-iASPP组中处于G0/G1期的细胞比例为74.65%±1.09%（F=388.901,P=0.000）,明显高于CON组（55.19%±1.02%）和shRNA-NC组（54.62%±0.88%）。侵袭实验显示:shRNA-iASPP组的侵袭细胞数目为56±4,比CON组（111±3）和shRNA-NC组（105±8）显著降低（F=84.965,P=0.000）。CCK-8实验结果表明:在紫杉醇作用下,shRNA-iASPP组细胞的存活率显著低于CON组和shRNA-NC组（F=634.841,P=0.000）。

结论 iASPP在HNSCC的发生、发展中起重要作用,有可能成为增强头颈鳞状细胞癌对紫杉醇化疗敏感性的有效分子治疗靶点。

关键词: 头颈鳞状细胞癌, iASPP, 化疗敏感性, 紫杉醇

Abstract:

Objective Our previous study has revealed that iASPP is elevated in human head and neck squamous cell carcinoma (HNSCC) and iASPP overexpression signifcantly correlates with tumor malignant progression and poor survival of HNSCC. This study investigated the function of iASPP playing in proliferation and invasion of HNSCC in vitro.

Methods HNSCC cell line Tu686 transfected with Lentiviral vector-mediated iASPP-specific shRNA and control shRNA were named the shRNA-iASPP group and shRNA-NC group, respectively. The non-infected Tu686 cells were named the CON group. CCK-8 assay, flow cytometry, transwell invasion assay were performed to detect the effects of iASPP inhibition in vitro.

Results Our results demonstrated that the proliferation of shRNA-iASPP cells at the time of 72 h (F=32.459, P=0.000), 96 h (F=51.407, P=0.000), 120 h (F=35.125, P=0.000) post-transfection, was significantly lower than that of shRNA-NC cells and CON cells. The apoptosis ratio of shRNA-iASPP cells was 9.42% ± 0.39% (F=299.490, P=0.000), which was significantly higher than that of CON cells (2.80% ± 0.42%) and shRNA-NC cells (3.18% ± 0.28%). The percentage of shRNA-iASPP cells in G0/G1 phase was 74.65% ± 1.09% (F=388.901, P=0.000), which was strikingly increased, compared with that of CON cells (55.19% ± 1.02%) and shRNA-NC cells (54.62% ± 0.88%). The number of invading cells was 56 ± 4 in the shRNA-iASPP group (F=84.965, P=0.000), which decreased significantly, compared with the CON group (111 ± 3) and the shRNA-NC group (105 ± 8). The survival rate of shRNA-iASPP cells administrated with paclitaxel was highly decreased, compared with CON cells and shRNA-NC cells (F=634.841, P=0.000).

Conclusion These results suggest iASPP may play an important role in progression and aggressive behavior of HNSCC and may be an efficient chemotherapeutic target for the treatment of HNSCC.

Key words: human head and neck squamous cell carcinoma, iASPP, chemosensitivity, paclitaxel

Liu Zhengzheng, Kuang Weilu, Zeng Wenjing, Xiao Jianyun, Tian Yongquan. Downregulation of iASPP Expression Suppresses Proliferation, Invasion and Increases Chemosensitivity to Paclitaxel of Head and Neck Squamous Cell Carcinoma In Vitro[J].Chinese Medical Sciences Journal, 2019, 34(3): 184-193.

图/表 9

参考文献 39.

1.	J-Fatokun F, Jayaratne R, Morawska L , et al. Corona ions from overhead transmission voltage powerlines: effect on direct current electric field and ambient particle concentration levels. Environ Sci Technol 2010; 44(1):526-31. doi: 10.1021/es9024063.
2.	Siegel R, Naishadham D, Jemal A . Cancer statistics, 2013. CA Cancer J Clin 2013; 63(1):11-30. doi: 10.3322/caac.21166.
3.	Jemal A, Siegel R, Xu J , et al. Cancer statistics, 2010. CA Cancer J Clin 2010; 60(5):277-300. doi: 10.3322/caac.20073.
4.	Hardisson D . Molecular pathogenesis of head and neck squamous cell carcinoma. Eur Arch Otorhinolaryngol 2003; 260(9):502-8. doi: 10.1007/s00405-003-0581-3.
5.	Chin D, Boyle GM, Theile DR , et al. Molecular introduction to head and neck cancer (HNSCC) carcinogenesis. Br J Plast Surg 2004; 57(7):595-602. doi: 10.1016/j.bjps.2004.06.010. doi: 10.1016/j.bjps.2004.06.010
6	Trigiante G, Lu X . ASPP [corrected] and cancer. Nat Rev Cancer 2006; 6(3):217-26. doi: 10.1038/nrc1818.
7.	Bergamaschi D, Samuels Y, O’Neil NJ , et al. iASPP oncoprotein is a key inhibitor of p53 conserved from worm to human. Nat Genet 2003; 33(2):162-7. doi: 10.1038/ng1070.
8.	Zhang X, Wang M, Zhou C , et al. The expression of iASPP in acute leukemias. Leuk Res 2005; 29(2):179-83. doi: 10.1016/j.leukres.2004.07.001.
9.	Lu B, Guo H, Zhao J , et al. Increased expression of iASPP, regulated by hepatitis B virus X protein-mediated NF-kappaB activation, in hepatocellular carcinoma. Gastroenterology 2010; 139(6):2183-94. doi: 10.1053/j.gastro.2010.06.049.
10.	Jiang L, Siu MK, Wong OG , et al. iASPP and chemoresistance in ovarian cancers: effects on paclitaxel-mediated mitotic catastrophe. Clin Cancer Res 2011; 17(21):6924-33. doi: 10.1158/1078-0432.CCR-11-0588.
11.	Wang L, Li Y, Li L , et al. Role of Kruppel-like factor 4 in regulating inhibitor of apoptosis-stimulating protein of p53 in the progression of gastric cancer. Oncol Lett 2018; 15(5):6865-72. doi: 10.3892/ol.2018.8203.
12.	Yin L, Lin Y, Wang X , et al. The family of apoptosis-stimulating proteins of p53 is dysregulated in colorectal cancer patients. Oncol Lett 2018; 15(5):6409-17. doi: 10.3892/ol.2018.8151.
13.	Chen J, Xie F, Zhang L , et al. iASPP is over-expressed in human non-small cell lung cancer and regulates the proliferation of lung cancer cells through a p53 associated pathway. BMC Cancer 2010; 10:694. doi: 10.1186/1471-2407-10-694.
14.	Zhang B, Xiao HJ, Chen J , et al. Inhibitory member of the apoptosis-stimulating protein of p53 (ASPP) family promotes growth and tumorigenesis in human p53-deficient prostate cancer cells. Prostate Cancer Prostatic Dis 2011; 14(3):219-24. doi: 10.1038/pcan.2011.25.
15.	Liu Z, Zhang X, Huang D , et al. Elevated expression of iASPP in head and neck squamous cell carcinoma and its clinical significance. Med Oncol 2012; 29(5):3381-8. doi: 10.1007/s12032-012-0306-9. doi: 10.1007/s12032-012-0306-9
16.	Livak KJ, Schmittgen TD . Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001; 25(4):402-8. doi: 10.1006/meth.2001.1262.
17.	Liu Y, Zhang X, Qiu Y , et al. Clinical significance of EphA2 expression in squamous-cell carcinoma of the head and neck. J Cancer Res Clin Oncol 2011; 137(5):761-9. doi: 10.1007/s00432-010-0936-2.
18.	Liu Z, Kuang W, Zhou Q , et al. TGF-beta1 secreted by M2 phenotype macrophages enhances the stemness and migration of glioma cells via the SMAD2/3 signalling pathway. Int J Mol Med 2018; 42(6):3395-403. doi: 10.3892/ijmm.2018.3923.
19.	Gan W, Zhao H, Li T , et al. CDK1 interacts with iASPP to regulate colorectal cancer cell proliferation through p53 pathway. Oncotarget 2017; 8(42):71618-29. doi: 10.18632/oncotarget.17794.
20.	Ma Y, Zhu B, Liu X , et al. iASPP overexpression is associated with clinical outcome in spinal chordoma and influences cellular proliferation, invasion, and sensitivity to cisplatin in vitro. Oncotarget 2017; 8(40):68365-80. doi: 10.18632/oncotarget.20190.
21.	Lu W, Yu T, Liu S , et al. FHL2 interacts with iASPP and impacts the biological functions of leukemia cells. Oncotarget 2017; 8(25):40885-95. doi: 10.18632/oncotarget.16617.
22.	Dong P, Xiong Y, Watari H , et al. Suppression of iASPP-dependent aggressiveness in cervical cancer through reversal of methylation silencing of microRNA-124. Sci Rep 2016; 6:35480. doi: 10.1038/srep35480.
23.	Li G, Wang R, Gao J , et al. RNA interference-mediated silencing of iASPP induces cell proliferation inhibition and G0/G1 cell cycle arrest in U251 human glioblastoma cells. Mol Cell Biochem 2011; 350(1-2):193-200. doi: 10.1007/s11010-010-0698-9. doi: 10.1007/s11010-010-0698-9
24.	Liu T, Li L, Yang W , et al. iASPP is important for bladder cancer cell proliferation. Oncol Res 2011; 19(3-4):125-30.
25.	Morris EV, Cerundolo L, Lu M , et al. Nuclear iASPP may facilitate prostate cancer progression. Cell Death Dis 2014; 5:e1492. doi: 10.1038/cddis.2014.442.
26.	Liang S, Gong X, Zhang G , et al. MicroRNA-140 regulates cell growth and invasion in pancreatic duct adenocarcinoma by targeting iASPP. Acta Biochim Biophys Sin (Shanghai) 2016; 48(2):174-81. doi: 10.1093/abbs/gmv127.
27.	Liang XG, Meng WT, Hu LJ , et al. MicroRNA-184 modulates human central nervous system lymphoma cells growth and invasion by targeting iASPP. J Cell Biochem 2017; 118(9):2645-53. doi: 10.1002/jcb.25856.
28.	Chen J, Xiao H, Huang Z , et al. MicroRNA124 regulate cell growth of prostate cancer cells by targeting iASPP. Int J Clin Exp Pathol 2014; 7(5):2283-90.
29.	Liu K, Zhao H, Yao H , et al. MicroRNA-124 regulates the proliferation of colorectal cancer cells by targeting iASPP. Biomed Res Int 2013; 2013:867537. doi: 10.1155/2013/867537.
30.	Zhao WH, Wu SQ, Zhang YD . Downregulation of miR-124 promotes the growth and invasiveness of glioblastoma cells involving upregulation of PPP1R13L. Int J Mol Med 2013; 32(1):101-7. doi: 10.3892/ijmm.2013.1365. doi: 10.3892/ijmm.2013.1365
31.	Zhao H, Peng R, Liu Q , et al. The lncRNA H19 interacts with miR-140 to modulate glioma growth by targeting iASPP. Arch Biochem Biophys 2016; 610:1-7. doi: 10.1016/j.abb.2016.09.014.
32.	Xiong Y, Sun F, Dong P , et al. iASPP induces EMT and cisplatin resistance in human cervical cancer through miR-20a-FBXL5/BTG3 signaling. J Exp Clin Cancer Res 2017; 36(1):48. doi: 10.1186/s13046-017-0520-6.
33.	Posner MR, Hershock DM, Blajman CR , et al. Cisplatin and fluorouracil alone or with docetaxel in head and neck cancer. N Engl J Med 2007; 357(17):1705-15. doi: 10.1056/NEJMoa070956.
34.	Rapidis AD, Trichas M, Stavrinidis E , et al. Induction chemotherapy followed by concurrent chemoradiation in advanced squamous cell carcinoma of the head and neck: final results from a phase II study with docetaxel, cisplatin and 5-fluorouracil with a four-year follow-up. Oral Oncol 2006; 42(7):675-84. doi: 10.1016/j.oraloncology.2005.12.006.
35.	Vermorken JB, Remenar E, van Herpen C , et al. Cisplatin, fluorouracil, and docetaxel in unresectable head and neck cancer. N Engl J Med 2007; 357(17):1695-704. doi: 10.1056/NEJMoa071028.
36.	Yu J, Li L, Huang C . Downregulation of inhibition of apoptosis-stimulating protein of p53 (iASPP) suppresses cisplatin-resistant gastric carcinoma in vitro. Med Sci Monit 2017; 23:5542-49.
37.	Jia Y, Peng L, Rao Q , et al. Oncogene iASPP enhances self-renewal of hematopoietic stem cells and facilitates their resistance to chemotherapy and irradiation. FASEB J 2014; 28(7):2816-27. doi: 10.1096/fj.13-244632. doi: 10.1096/fj.13-244632
38.	Cao L, Huang Q, He J , et al. Elevated expression of iASPP correlates with poor prognosis and chemoresistance/radioresistance in FIGO Ib1-IIa squamous cell cervical cancer. Cell Tissue Res 2013; 352(2):361-9. doi: 10.1007/s00441-013-1569-y. doi: 10.1007/s00441-013-1569-y
39.	Liu H, Wang M, Diao S , et al. siRNA-mediated down-regulation of iASPP promotes apoptosis induced by etoposide and daunorubicin in leukemia cells expressing wild-type p53. Leuk Res 2009; 33(9):1243-8. doi: 10.1016/j.leukres.2009.02.016.

优先出版

当期目次

过刊浏览

虚拟专题

作者中心

评阅者中心