Chen Zhuanpeng, Cao Jie, Yang Ping, Liu Zhenbang, Wei Jianchang, Chen Huacui, Qiu Xubin, Hu He
Department of Gastrointestinal Surgery, Affiliated Guangzhou Municipal People's Hospital, Guangzhou Medical University, Guangzhou 510180, China.
Zhonghua Wei Chang Wai Ke Za Zhi. 2016 Jun;19(6):695-701.
To investigate the effect and its possible mechanism of interleukin-17 (IL-17) on invasion and metastasis of human colon cancer cells.
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 μg/L IL-17+SW480 cells), and LOVO experiment group (50 μg/L IL-17+LOVO cells). Cell growth was measured by CCK-8 assay. The proliferation rate(%)=[(Aexperiment group-Ablank)/(Acontrol 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.
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).
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.
探讨白细胞介素-17（IL-17）对人结肠癌细胞侵袭和转移的影响及其可能机制。
将IL-17加入人结肠癌细胞SW480和LOVO的培养基中。细胞分为4组：SW480对照组（SW480细胞）、LOVO对照组（LOVO细胞）、SW480实验组（50μg/L IL-17 + SW480细胞）和LOVO实验组（50μg/L IL-17 + LOVO细胞）。采用CCK-8法检测细胞生长情况。增殖率(%) = [(实验组A - 空白组A)/(对照组A - 空白组A)]×100%。采用transwell法检测细胞侵袭和迁移能力。采用实时荧光定量PCR检测VEGF和MMP-9的mRNA表达。采用蛋白质免疫印迹法检测STAT3、p-STAT3、VEGF和MMP-9的蛋白表达。采用酶联免疫吸附测定法（ELISA）检测上清液中VEGF和MMP-9的蛋白含量。
培养24、48和72小时后，CCK-8法检测显示，SW480细胞在实验组中的增殖率分别为1.18%±0.07%、1.42%±0.09%和1.62%±0.08%；LOVO细胞在实验组中的增殖率分别为1.13%±0.02%、1.32%±0.05%和1.73%±0.02%。Transwell实验表明，与IL-17共培养24小时后，实验组（SW480：34.00±0.45，LOVO：41.60±0.51）的侵袭细胞数高于相应对照组（SW480：4.53±0.14；LOVO：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）。实验组（SW480：36.40±0.51，LOVO：46.40±0.68）的迁移细胞数高于相应对照组（SW480：7.83±0.69；LOVO：6.67±0.48），差异有统计学意义（SW480：t = -51.542，P = 0.003；LOVO：t = -49.265，P = 0.005）。实时荧光定量PCR结果显示，与IL-17共培养24小时后，实验组（SW480：VEGF：1.53±0.12，MMP-9：2.44±0.23；LOVO：VEGF：2.96±0.35，MMP-9：3.38±0.55）中VEGF和MMP-9的mRNA相对表达水平高于对照组（均为1），差异有统计学意义（VEGF：t = 3.799，P = 0.043；MMP-9：t = 5.254，P = 0.039）。蛋白质免疫印迹法表明，与IL-17共培养24小时后，实验组中STAT3、p-STAT3、VEGF和MMP-9的蛋白相对表达水平显著高于对照组（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检测显示，实验组上清液中VEGF和MMP-9的含量（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）高于对照组（SW480：VEGF：4 456.32±87.56，MMP-9：18.57±2.44。LOVO：VEGF：8 122.38±108.66，MMP-9：163.
