摘要
目的探讨白介素17(IL-17)对人结肠癌细胞株SW480和LOVO细胞增殖、侵袭和迁移能力的作用,以及可能的机制。方法根据外源性人IL-17是否作用于人结肠癌细胞株SW480和LOVO,分为4组细胞:SW480对照组(SW480细胞)、LOVO对照组(LOVO细胞)、SW480实验组(50μg/LIL-17处理SW480细胞)和LOVO实验组(50μg/LIL-17处理LOVO细胞)。CCK-8法检测各组细胞增殖活性,细胞增殖率(%)=[(A实验组-A空白组)/(A对照组-A空白组)]×100%;Transwel]实验检测各组细胞侵袭和迁移能力,实时定量聚合酶链反应(realtime.PCR)检测各组细胞血管内皮生长因子(VECF)、基质金属蛋白酶9(MMP-9)mRNA的表达,蛋白质印迹(western blot)检测各组细胞信号传导与转录激活因子3(STAT3)、磷酸化STAT3(p-STAT3)、VEGF和MMP-9蛋白的表达,酶联免疫吸附法(ELISA)检测各组细胞上清液中VEGF、MMP-9蛋白的含量。结果培养24h、48h和72h后,实验组SW480细胞的增殖率分别为1.18%±0.07%、1.42%±0.09%和1.62%±0.08%,LOVO细胞的增殖率分别为1.13%±0.02%、1.32%±0.05%和1.73%±0.02%。Transwell实验显示,IL-17作用24h后.SW480实验组和LOVO实验组侵袭细胞个数分别为34.00±0.45和41.60±0.51,多于对照组(分别为4.53±0.14和3.67±0.33),差异有统计学意义(SW480:t=76.026,P=0.001;LOVO:t=-81.580,P=0.005);SW480实验组和LOVO实验组迁移细胞个数分别为36.40±0.51和46.40±0.68,多于对照组(分别为7.83±0.69和6.67±0.48),差异有统计学意义(SW480:t=-51.542,P=O.003;LOVO:t=-49.265,P=0.005)。实时定量聚合酶链反应结果显示,IL-17作用24h后,SW480实验组细胞VEGF、MMP-9mRNA相对表达量分别为1.53±0.12和2.44±0.23.高于SW480对照组(均为1.00),差异均有统计学意义(VEGF:t=3.211,P=0.027;MMP-9:t=4.306,P=0.025);LOVO实验组细胞VEGF、MMP-9mRNA相对表达量分别为2.96±0.35和3.38±0.55.高于LOVO对照组(均为1.00),差异均有统计学意义(VEGF:t=3.799,P=0.043;MMP-9:t=5.254,P=0.039)。Western blot实验表明,IL-17作用24h后,SW480实验组STAT3、P—STAT3、VEGF和MMP-9蛋白的相对表达量明显高于对照组(STAT3:t=3.233,P=0.023;p-STAT3:t=3.954,P=0.032;VEGF:t=3.201,P=0.025;MMP-9:t=3.154,P=0.029);LOVO实验组STAT3、p-STAT3、VECF和MMP-9蛋白的相对表达量明显高于对照组(STAT3:t=3.788,P=0.012:p-STAT3:t=2.662,P=0.040:VEGF:t=4.118,P=0.035;MMP-9:t=4.268,P=0.030)。ELISA实验结果显示,SW480细胞实验组上清液中VEGF和MMP-9的含量分别为5491.41±63.22和21.43±1.35,均高于对照组(分别为4456.32±87.56和18.57±2.44),差异具有统计学意义(VEGF:t=6.993,P=0.037;MMP.9:t=5.587,P=0.040)。LOVO细胞实验组VEGF和MMP-9的含量分别为8631.46±129.59和178.32±3.20.亦高于对照组(分别为8122.38±108.66和163.22±6.89),差异具有统计学意义(VEGF:t=7.013,P=0.044;MMP-9:t=6.762,P=0.043)。结论IL-17能上调SW480和LOVO人结肠癌细胞中STAT3和p-STAT3蛋白的表达,激活STAT3信号转导通路,从而上调VEGF和MMP-9蛋白的表达,增强SW480和LOVO结肠癌细胞的侵袭和迁移能力。
Objective To investigate the effect and its possible mechanism of interleukin-17 (IL-17) on invasion and metastasis of human colon cancer cells. Methods IL-17 was added into the culture media of human colon cancer cells SW480 and LOVO. Cells were divided into 4 groups: SW480 control group (SW480 cells), LOVO control group (LOVO cells), SW480 experiment group (50 Ixg/ L IL-17+SW480 cells), and LOVO experiment group (50 p,g/L IL-17+LOVO cells). Cell growth was measured by CCK-8 assay. The proliferation rate (%) =[ (A experiment group-Ablank)/(A control group- Ablank)] ×100%). The ability of cell invasion and migration was measured by transwell assay. Real time-PCR was used to detect mRNA expression of VEGF and MMP-9. Western blot was performed to detect protein expression of STAT3, p-STAT3, VEGF and MMP-9. Enzyme-linked immunosorbent assay (ELISA) was applied to measure the protein content of VEGF and MMP-9 in the supernatant. Results After cultivation for 24, 48 and 72 hours, CCK-8 assay revealed that the proliferation rate of SW480 was 1.18% ± 0.07%, 1.42%± 0.09%, and 1.62%± 0.08%; the proliferation rate of LOVO was 1.13% ± 0.02%, 1.32%± 0.05% and 1.73%±0.02% in experiment group. Transwell experiments showed that after cultivation with IL-17 for 24 hours, number of invasion cell in experimental groups (SW480: 34.00 ± 0.45, LOVO: 41.60 ±0.51 ) was higher as compared to corresponding control groups (SW480: 4.53 ± 0.14; LOVO: 3.67 ± 0.33) with significant differences (SW480: t = -76.026, P= 0.001; LOVO: t =-81.580, P= 0.005). The number of migration cell in experimental groups (SW480:36.40 ± 0.51, LOVO: 46.40 ± 0.68) was higher as compared to corresponding control groups (SW480:7.83 ± 0.69; LOVO: 6.67 ±0.48) with significant differences (SW480: t = -51.542, P = 0.003; LOVO: t = -49.265, P = 0.005). Real-time PCR results revealed that after cultivation with IL-17 for 24 hours, VEGF and MMP-9 mRNA relative expression levels in experimental groups (SW480: VEGF: 1.53 ± 0.12, MMP-9:2.44±0.23; LOVO: VEGF: 2.96±0.35, MMP-9:3.38 ± 0.55) were higher than those in control groups (both 1) with significant differences (VEGF: t=3.799, P=0.043; MMP-9: t=5.254, P=0.039). Western blot illustrated that after cultivation with IL-17 for 24 hours, STAT3, p-STAT3, VEGF and MMP-9 proteins relative expression levels in experimental groups were significantly higher that those in control groups (SW480: STAT3 : t = 3.233, P = 0.023 ; p-STAT3 : t = 3.954, P = 0.032 ; VEGF: t=3.201, P=0.025; MMP-9: t=3.154, P=0.029; LOVO: STAT3: t=3.788, P=0.012; p-STAT3: t = 2.662, P= 0.040; VEGF: t = 4.118, P= 0.035; MMP-9: t = 4.268, P= 0.030). ELISA indicated that content of VEGF and MMP-9 in the supernatant of experimental groups (SW480: VEGF 5 491.41 ± 63.22, MMP-9:21.43 ± 1.35. LOVO: VEGF: 8 631.46 ± 129.59, MMP-9:178.32 ± 3.20) were higher than those in control groups (SW480:VEGF:4 456.32 ± 87.56, MMP-9:18.57 ±2.44. LOVO: VEGF: 8 122.38 ±108.66, MMP-9:163.22 ± 6.89) with significant differences (SW480: VEGF: t=6.993,P=0.037; MMP-9: t=5.587, P=0.040. LOVO: VEGF: t=7.013, P=0.044; MMP-9: t = 6.762, P= 0.043). Conclusion IL-17 may be able to activate STAT3 signal transduction pathway in vitro through up-regulation of VEGF and MMP-9 expression, thereby enhancing the invasion and migration of colon cancer SW480 and LOVO cells.
出处
《中华胃肠外科杂志》
CAS
CSCD
北大核心
2016年第6期695-701,共7页
Chinese Journal of Gastrointestinal Surgery
基金
国家自然科学基金(81272556)
广东省科技基金(2010B060900016)
关键词
白介素17
结肠肿瘤
信号传导与转录激活因子3
侵袭迁移
IL-17
Colon neoplasms
Signal transduction and activator of transcription 3
Invasion and Migration