COX-2在口腔鳞状细胞癌增殖与侵袭中的作用研究

doi:10.11958/20253464

摘要/Abstract

摘要：

目的探讨环氧合酶-2（COX-2）在口腔鳞状细胞癌（OSCC）细胞增殖、迁移、侵袭及体内成瘤中的作用。方法选用海南省肿瘤医院口腔科术后5例正常口腔黏膜组织及5例口腔OSCC组织，采用免疫组化检测COX-2在二者之间的表达差异；使用塞来昔布及COX-2 shRNA抑制SCC9细胞COX-2表达，Western blot验证抑制效率。通过CCK-8、克隆形成实验评估细胞增殖能力；细胞划痕实验与侵袭实验检测细胞迁移及侵袭能力；构建SCC9裸鼠皮下移植瘤模型，观察COX-2沉默对肿瘤生长的影响。结果免疫组化结果显示，COX-2在正常口腔黏膜组织中多为阴性或弱阳性表达，而在OSCC组织中呈明显强阳性表达。与Control组相比，Celecoxib处理及COX-2 shRNA转染均可下调SCC9细胞COX-2蛋白表达水平，降低细胞OD₄₅₀值、克隆数、划痕愈合率和穿膜细胞数（P<0.05）。裸鼠移植瘤实验显示，随着成瘤时间延长，COX-2沉默组肿瘤体积减小，最终测量体质量低于空载体组（P<0.05）。结论 COX-2在OSCC中高表达并参与肿瘤的增殖、迁移、侵袭及体内生长，抑制COX-2可削弱OSCC细胞的恶性生物学行为。

关键词: 口腔肿瘤, 癌, 鳞状细胞, 环氧化酶2, 细胞增殖, 细胞运动

Abstract:

Objective To explore the role of cyclooxygenase-2 (COX-2) in the proliferation, migration, invasion and tumorigenesis in vivo of oral squamous cell carcinoma (OSCC) cells. Methods Tissue samples, including five cases of normal oral mucosa and five cases of OSCC, were collected postoperatively from the Department of Stomatology, Hainan Cancer Hospital. Immunohistochemistry was performed to detect the expression of COX-2 in both groups. Celecoxib and COX-2 shRNA were used to inhibit the expression of COX-2 in SCC9 cells, and the inhibition efficiency was verified by Western blot assay. The proliferation ability of cells was evaluated by CCK-8 and colony formation assays. The migration and invasion abilities of cells were detected by scratch and invasion assays. A subcutaneous xenograft tumor model of SCC9 cells in nude mice was established to observe the effect of COX-2 silencing on tumor growth. Results Immunohistochemistry showed that COX-2 was mostly negative or weakly positive in normal oral mucosa tissue, but strongly positive in OSCC tissue. Compared with the control group, both Celecoxib treatment and COX-2 shRNA transfection could down-regulate the expression of COX-2 protein in SCC9 cells (P<0.05), and reduce the OD₄₅₀ value of cells, the number of clones, the scratch healing rate and the number of transmembrane cells. The xenograft tumor experiment in nude mice showed that with the extension of tumor formation time, the tumor volume in the COX-2 silencing group decreased, and the final measured body weight was lower than that in the empty vector group (P<0.05). Conclusion COX-2 is highly expressed in OSCC and participates in the proliferation, migration, invasion and in vivo growth of tumors. The inhibition of COX-2 can weaken the malignant biological behavior of OSCC cells.

Key words: mouth neoplasms, carcinoma, squamous cell, cyclooxygenase 2, cell proliferation, cell movement

中图分类号:

R739.8

图/表 8

图1 COX-2在正常口腔黏膜及口腔鳞状细胞癌组织中的表达（免疫组化，×200）

Fig.1 Expression of COX-2 in normal oral mucosa and oral squamous cell carcinoma tissue (IHC, ×200)

图2 各组COX-2蛋白表达情况 A：Celecoxib对COX-2表达影响，a—d分别为Control组、Celecoxib 10 μg/L组、Celecoxib 20 μg/L组、Celecoxib 50 μg/L组；B：RNA干扰对COX-2表达的影响。

Fig.2 The expression of COX-2 in each group

表1 各组细胞在不同时间点的OD450值比较（n=3，$\bar{x}±s$）

Tab.1 Comparison of OD450 value of cells between the five groups

组别	24 h	48 h	72 h	96 h
Control组	0.35±0.01	0.59±0.07	0.84±0.02	1.08±0.03
Empty Vector组	0.34±0.02	0.57±0.02	0.82±0.03	1.07±0.00
Celecoxib 20 μg/L组	0.35±0.05	0.45±0.00^ab	0.70±0.02^ab	0.83±0.01^ab
Celecoxib 50 μg/L组	0.31±0.03	0.46±0.03^ab	0.56±0.01^abc	0.72±0.03^abc
COX-2 shRNA组	0.32±0.05	0.46±0.04^ab	0.64±0.04^abcd	0.73±0.02^abc
F	0.732	8.903^＊＊	7.301^＊＊	14.554^＊＊

表2 各组细胞克隆形成、划痕愈合及穿膜能力比较（n=3，$\bar{x}±s$）

Tab.2 Comparison of colony formation, wound healing and Transwell invasion ability between the five groups

组别	克隆数/个	划痕愈合率/%	穿膜细胞数/个
Control组	223.09±10.94	64.51±13.09	126.33±11.28
Celecoxib 20 μg/L组	145.04±5.06^a	46.24±6.13^a	82.64±6.15^a
Celecoxib 50 μg/L组	26.99±13.66^ab	27.41±3.19^ab	54.18±5.02^ab
Empty Vector组	197.43±2.17	66.55±3.90	121.47±9.36
COX-2 shRNA组	45.55±5.55^ac	33.96±2.77^ac	49.37±4.88^ac
F	318.073^＊＊	19.218^＊＊	65.220^＊＊

图3 各组SCC9细胞克隆形成能力比较

Fig.3 Comparison of clone formation ability of SCC9 cells between the five groups

图4 各组SCC9细胞迁移能力比较

Fig.4 Comparison of migration ability of SCC9 cells between the five groups

图5 各组SCC9细胞侵袭能力比较

Fig.5 Comparison of the invasion ability of SCC9 cells between the five groups

表3 2组不同时间点裸鼠移植瘤体积比较（n=5，mm3，$\bar{x}±s$）

Tab.3 Comparison of tumor volumes in nude mice at different time points between the two groups

组别	0周	1周	2周	3周	4周	5周	6周
Empty Vector组	52.18±9.24	130.46±18.59	289.74±35.42	559.87±62.28	919.65±85.73	1 270.52±110.36	1 519.74±135.82
COX-2shRNA组	48.92±8.26	95.38±15.63	184.56±28.19	329.42±40.74	520.28±55.42	689.84±70.58	610.29±58.46
t	0.588	3.230^＊	5.195^＊＊	12.401^＊＊	16.230^＊＊	17.816^＊＊	31.632^＊＊

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