Effects of Cyclooxygenase-2 Gene Silencing on the Arachidonic Acid Metabolism Pathway of Esophageal Squamous Cell Carcinoma Cells
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摘要:
目的 探讨siRNA沉默环氧合酶-2（COX-2）对食管鳞癌（ESCC）细胞增殖的影响，观察花生四烯酸（AA）代谢酶COX-2和5-脂氧合酶（5-LOX）、其下游产物PGE2和LTB4及细胞凋亡相关基因表达的变化，寻找低浓度COX-2酶抑制剂引起AA代谢转向5-LOX途径并促进ESCC细胞增殖的解决方案。 方法 设空白对照、脂质体对照、随机序列siRNA和COX-2 siRNA组，筛选高效COX-2 siRNA序列作用于ESCC细胞株TE-1及Eca109，四唑单钠法检测细胞增殖、RT-PCR和Western blot检测mRNA及蛋白、ELISA法检测PGE2和LTB4，流式细胞技术检测细胞周期。 结果 与空白对照组相比，COX-2 siRNA转染后，TE-1及Eca109细胞出现增殖抑制（抑制率分别为45.86%和48.99%，均P < 0.05）；COX-2 mRNA、蛋白表达及PGE2下降（P < 0.05），5-LOX表达及LTB4无明显变化（P>0.05）；G1期细胞比例分别为63.16 %和68.15 %（空白对照组分别为58.93%和33.02%，P < 0.05）；Bcl-2 mRNA及蛋白表达下降而Caspase-9、Bax表达上调（P < 0.05）。 结论 高效COX-2抑制可以避免COX-2低水平抑制引起的AA向5-LOX途径代谢分流，从而有效实现ESCC细胞增殖抑制。 Abstract:Objective In a previous study, we found that low doses of cyclooxygenase-2 (COX-2) selective inhibitors could induce the proliferation of esophageal squamous cell carcinoma (ESCC) cells, which can be attributed to the activation of a 5-lipoxygenase (5-LOX) shunt by slight COX-2 inhibition. The objective of the present study was to determine whether highly effective COX-2 siRNA inhibition can avoid this shunt. Methods TE-1 and Eca109 (ESCC) cells were divided into blank control, liposome transfection, random sequence siRNA, and COX-2 siRNA groups. Cell proliferation was assessed using Cell Counting Kit-8 assay. Protein and mRNA expressions were determined using Western blot analysis and RT-PCR, respectively. Prostaglandin E2 and leukotriene B4 (LTB4) levels were measured by enzyme-linked immunosorbent assay. A flow cytometer was used for cell cycle measurement. Results Compared with the blank controls, COX-2 siRNA-transfected TE-1 and Eca109 cells showed 79% and 73% inhibition of COX-2 expression, respectively, as well as 45.86% and 48.99% inhibition of cell proliferation, respectively (P < 0.05). The expression of 5-LOX remained unchanged (P>0.05), and prostaglandin E2 and LTB4 levels were highly in accordance with alterations in COX-2 and 5-LOX expressions, respectively. The percentages of cells in G1 stage increased significantly. Bcl-2 expression decreased, whereas the expressions of caspase-9 and Bax increased in the two ESCC cells after COX-2 siRNA transfection (P < 0.05). Conclusions Highly effective inhibition of COX-2 expression may prevent the activation of 5-LOX and the following up-regulation of LTB4, which is a cell proliferation factor. These suggest that only high-dose COX-2 selective inhibitors with significant COX-2 inhibitory effects can achieve anti-cancer effects in ESCCs. -
Key words:
- RNA interference /
- Cyclooxygenase-2 /
- 5-Lipoxygenase /
- Esophageal squamous cell carcinoma /
- Apoptosis
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图 1 siRNA干预与COX-2、5-LOX、Bcl-2、Caspase-9及Bax mRNA及蛋白在TE-1（A）及Eca109（B）细胞的表达
1：空白对照；2：脂质体对照；3：随机序列siRNA；4：COX-2 siRNA
Figure 1. mRNA and protein expressions of COX-2, 5-LOX, Bcl-2, caspase-9, and Bax in TE-1(A)and Eca109(B)cells treated with siRNA
表 1 引物序列及退火温度
Table 1. Sequences of the selected primer pair and annealing temperature in reactive polymerase chain reaction
表 2 COX-2 siRNA转染后TE-1与Eca109细胞COX-2、5-LOX、Bcl-2、Caspase-9和Bax mRNA及蛋白的表达
Table 2. mRNA and protein expressions of COX-2, 5-LOX, Bcl-2, caspase-9, and Bax in TE-1 and Eca109 cells treated with COX-2 siRNA
表 3 COX-2 siRNA转染后TE-1及Eca109细胞PGE2和LTB4水平的变化（x±s，n=3）
Table 3. PGE2 and LTB4 levels in TE-1 and Eca109 cells treated with COX-2 siRNA
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