CYP3A5在口腔鳞状细胞癌组织中的表达及作用

doi:10.3969/j.issn.1005-4979.2022.03.004

摘要/Abstract

摘要：

目的：探究CYP3A5在口腔鳞状细胞癌（oral squamous cell carcinoma, OSCC）组织及细胞中的表达及其对OSCC细胞生物学行为能力的影响。方法：采用实时荧光定量聚合酶链反应（real-time quantitative polymerase chain reaction, RT-qPCR）法和蛋白质免疫印迹（Western blotting）法分别检测OSCC组织及细胞中CYP3A5基因和蛋白的表达，通过CYP3A5 siRNA敲除降低CYP3A5表达水平，观察CYP3A5基因对OSCC细胞增殖、侵袭、迁移等能力的影响。通过免疫组织化学（immunohistochemistry，IHC）法检测OSCC及癌旁组织中CYP3A5的表达水平。结果：与癌旁组织相比，OSCC组织中的CYP3A5呈明显低表达（P<0.05）；与正常口腔上皮细胞系HOK相比，OSCC细胞系SCC-9、SCC-25、CAL-27中CYP3A5的表达水平明显偏低（P<0.05）。抑制CYP3A5基因表达能明显促进SCC-9、SCC-25、CAL-27的增殖、侵袭和迁移能力（P<0.05）。免疫组织化学分析结果显示，CYP3A5在OSCC癌组织中呈显著低水平。结论：CYP3A5在OSCC组织和细胞中呈明显低表达，对于促进OSCC的增殖、侵袭和迁移具有重要意义。

关键词: 口腔鳞状细胞癌, CYP3A5, 增殖, 侵袭

Abstract:

Objective: To investigate the expression level of CYP3A5 in oral squamous cell carcinoma (OSCC) tissues and cells and its effects on the biological behavior of OSCC. Methods: Real-time quantitative polymerase chain reaction (RT-qPCR) and Western blotting were used to detect the genetic and protein expression of CYP3A5 in OSCC tissues and cells respectively, CYP3A5 siRNA was used to decrease the expression level of CYP3A5, and the effects of CYP3A5 gene on the proliferation, invasion and migration of OSCC cells were observed. The expression levels of CYP3A5 in OSCC and adjacent tissues were detected by immunohistochemistry(IHC). Results: Compared with adjacent cancer tissues, the genetic expression level of CYP3A5 was significantly lower in OSCC tissues(P<0.05). Compared with normal oral epithelial cell line HOK, the expression levels of CYP3A5 gene in OSCC cell lines SCC-9, SCC-25, and CAL-27 were significantly lower (P<0.05). Inhibiting the expression level of CYP3A5 gene significantly promoted the proliferation, invasion and migration capability of SCC-9, SCC-25, and CAL-27 cell lines(P<0.05). Immunohistochemical analysis showed that CYP3A5 demonstrated weaker signal in OSCC(P<0.05). Conclusion: The expression level of CYP3A5 was significantly lower in OSCC tissues and cells, which was of great significance for promoting the proliferation, invasion and migration of OSCC.

Key words: oral squamous cell carcinoma, CYP3A5, proliferation, invasion

陈伟鸿, 熊洁, 查骏. CYP3A5在口腔鳞状细胞癌组织中的表达及作用[J]. 《口腔颌面外科杂志》, 2022, 32(3): 158-163.

CHEN Weihong, XIONG Jie, ZHA Jun. Expression and effect of CYP3A5 gene in oral squamous cell carcinoma[J]. 《Journal of Oral and Maxillofacial Surgery》, 2022, 32(3): 158-163.

https://journal06.magtech.org.cn/Jweb_joms/CN/Y2022/V32/I3/158

图/表 7

表1 RT-qPCR引物序列

Table 1 The primer sequences of the genes used for RT-qPCR

基因名称	引物序列（5′ - 3′）
CYP3A5	上游引物： TGTTATTCTGTCTTCACAAATCGAA 下游引物： CCTCAAGTTTCTCACCAATACATCT
GADPH	上游引物： AAGAAGGTGGTGAAGCAGG 下游引物： GAAGGTGGAAGAGTGGGAGT

图1 OSCC组织及细胞系中CYP3A5的基因及蛋白表达水平 A. CYP3A5基因在10例OSCC组织中呈低表达水平，***代表P<0.001，与癌旁组织比较；B.CYP3A5基因在SCC-9、SCC-25、CAL-27细胞中呈低表达水平，**代表P<0.01、***代表P<0.001，与HOK细胞比较；C. CYP3A5蛋白在OSCC组织中呈低表达水平；D. CYP3A5蛋白在SCC-9、SCC-25、CAL-27细胞中呈低表达水平。

Figure 1 Genetic and protein expression levels of CYP3A5 in OSCC tissues and cell lines

图2 CYP3A5 siRNA的转染效率及细胞株筛选 A. 转染CYP3A5 siRNA-1后SCC-9、SCC-25、CAL-27细胞中CYP3A5基因的表达水平；B. 转染CYP3A5 siRNA-2后SCC-9、SCC-25、CAL-27细胞中CYP3A5基因的表达水平。*代表P<0.05、**代表P<0.01，与对照组比较。

Figure 2 Transfection efficiency of CYP3A5 siRNA and cell line screening

图3 抑制CYP3A5基因表达对 OSCC细胞系增殖的影响 A. 抑制CYP3A5基因表达在48、72 h后可促进SCC-9细胞增殖；B. 抑制CYP3A5基因表达在48、72 h后可促进SCC-25细胞增殖；C. 抑制CYP3A5基因表达在48、72 h后可促进CAL-27细胞增殖。*代表P<0.05、**代表P<0.01，与对照组比较。

Figure 3 Effect of inhibiting of the expression of CYP3A5 gene on the proliferation of OSCC cell lines

图4 抑制CYP3A5基因的表达促进OSCC细胞侵袭（×40） A、B. 抑制CYP3A5基因表达促进SCC-9细胞侵袭及统计结果；C、D. 抑制CYP3A5基因表达促进SCC-25细胞侵袭及统计结果；E、F. 抑制CYP3A5基因表达促进CAL-27细胞侵袭及统计结果。*代表P<0.05、**代表P<0.01，与对照组比较。

Figure 4 Inhibition of the expression of CYP3A5 gene promoted the invasion of OSCC cell lines（×40）

图5 抑制CYP3A5的表达促进OSCC细胞迁移（×40） A、B. 抑制CYP3A5基因表达促进SCC-9细胞迁移及统计结果；C、D. 抑制CYP3A5基因表达促进SCC-25细胞迁移及统计结果；E、F. 抑制CYP3A5基因表达促进CAL-27细胞迁移及统计结果。*代表P<0.05、**代表P<0.01，与对照组比较。

Figure 5 Inhibiting of the expression of CYP3A5 gene promoted the migration of OSCC cell lines（×40）

图6 免疫组织化学法分析CYP3A5在OSCC组织中的表达水平（×40） A. 显微镜下OSCC组织及癌旁组织免疫组织化学染色情况；B. CYP3A5免疫组织化学染色阳性面积统计学结果。**代表P<0.01，与癌旁组织比较。

Figure 6 IHC analysis of CYP3A5 staining expression level in OSCC tissues（×40）

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