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目的探讨环氧合酶-2(COX-2)反义核酸对膀胱癌细胞恶性表型的抑制作用。方法采用脂质体介导方法,分别构建转染COX-2反义真核表达载体和空载体的膀胱癌细胞系5637-AS细胞和5637-P细胞,RT-PCR及W estern b lot分析转染细胞COX-2 mRNA及蛋白表达水平。MTT比色实验、Boyden侵袭小室法和裸鼠成瘤实验分别检测转染细胞体外增殖速度、体外侵袭力和体内成瘤性。结果RT-PCR结果显示,5637-AS细胞COX-2/磷酸甘油醛脱氢酶(GAPDH)mRNA比值(0.65)明显低于5637(0.92)和5637-P细胞(0.90);W estern b lot提示5637-AS细胞COX-2/β-actin比值(0.29)明显低于5637细胞(0.46)和5637-P细胞(0.44)。MTT比色实验显示5637-AS细胞较5637-P、5637细胞生长速度减慢,三者倍增时间分别为3.6 d、2.9 d和2.9 d。体外侵袭实验结果表明,5637-AS侵袭细胞数显著低于5637细胞及5637-P细胞(18.20±5.97比45.40±8.12,18.20±5.97比44.10±6.47,P<0.01)。裸鼠成瘤实验中,5637-P、5637细胞接种裸鼠后第5天肿瘤长出,而5637-AS细胞第7天肿瘤长出;30 d后5637-AS细胞接种组瘤体重量(392.15 mg±33.58 mg)明显小于5637细胞(738.26 mg±66.32 mg),和5637-P细胞接种组(698.53 mg±88.76 mg),P<0.01。结论膀胱癌细胞中COX-2过表达与细胞的恶性表型相关,反义技术抑制COX-2表达可以逆转膀胱癌细胞的恶性表型。
Objective To investigate the inhibitory effect of cyclooxygenase-2 (COX-2) antisense oligonucleotide on the malignant phenotype of bladder cancer cells. Methods The bladder cancer cell lines 5637-AS and 5637-P transfected with COX-2 antisense eukaryotic expression vector and empty vector were constructed by liposome-mediated method. RT-PCR and Western blot analysis Dye cells COX-2 mRNA and protein expression levels. MTT colorimetric assay, Boyden invasion chamber method and nude mouse tumorigenicity assay were used to detect the proliferation rate, in vitro invasiveness and in vivo tumorigenicity of transfected cells respectively. Results The RT-PCR results showed that the ratio of COX-2 / GAPDH mRNA in 5637-AS cells was significantly lower than that in 5637 cells (0.92) and 5637-P cells (0.90) The COX-2 / β-actin ratio (0.29) in 5637-AS cells was significantly lower than that in 5637 cells (0.46) and 5637-P cells (0.44). Compared with 5637-P and 5637 cells, MTT colorimetric assay showed that the growth rate of 5637-AS cells was slower than that of 5637-P cells, with the doubling time of 3.6 d, 2.9 d and 2.9 d respectively. In vitro invasion assay showed that the number of 5637-AS-invasive cells was significantly lower than that of 5637 cells and 5637-P cells (18.20 ± 5.97 vs. 45.40 ± 8.12, 18.20 ± 5.97 vs. 44.10 ± 6.47, P <0.01). In nude mice, 5637-P and 5637 cells grew on day 5 after inoculation in nude mice, while 5637-AS cells grew on day 7; after 30 days, 5637-AS cells were inoculated on tumor weight (392.15 mg ± 33.58 mg) was significantly less than 5637 cells (738.26 mg ± 66.32 mg) and 5637-P cells (698.53 mg ± 88.76 mg), P <0.01. Conclusion Overexpression of COX-2 in bladder cancer cells is associated with the malignant phenotype of the cells. Antisense technology can inhibit the malignant phenotype of bladder cancer cells by inhibiting the expression of COX-2.