白细胞介素22通过活化STAT3通路促进胰腺癌细胞增殖、迁移和侵袭

Interleukin 22 promotes proliferation,migration and invasion of pancreatic cancer cells by activating STAT3 pathway

目的:研究白细胞介素-22(interleukin-22,IL-22)对胰腺癌细胞增殖、迁移和侵袭能力的影响并探索其可能机制。探索IL-22对不同恶性程度的胰腺癌细胞系促癌效果差异。方法:选择恶性程度较低的SW1990和恶性程度较高的CAPAN-2两种胰腺导管癌细胞做平行实验。MTT法检测IL-22对胰腺癌细胞增殖的影响;细胞划痕实验观察IL-22对胰腺癌细胞迁移能力的影响;Transwell小室侵袭实验检测IL-22对胰腺癌细胞侵袭能力的影响;RT-PCR检测经IL-22处理前后,胰腺癌细胞中血管内皮生长因子(vascular endothelial growth factor,VEGF)、高迁移率族蛋白B1(high mobility group protein B1,HMGB1)、基质金属蛋白酶9(matrix metalloproteinase 9,MMP9)的mRNA表达差异;Western blot实验检测IL-22处理前后,细胞中信号转导及转录激活蛋白3(signal transducer and activator of transcription 3,STAT3)、p-STAT3、VEGF、HMGB1、MMP9的蛋白表达差异。以上实验均进行两种胰腺癌细胞系之间的平行比较。结果:MTT实验中,在不同浓度的IL-22作用下,胰腺癌细胞光密度(optical density,OD)值随着浓度增加逐渐增高,CAPAN-2细胞的增长率大于SW1990细胞的增长率;划痕实验显示,在IL-22作用后24 h和48 h,划痕相对宽度分别逐渐减小,CAPAN-2细胞的24 h变化率和48 h变化率均大于SW1990细胞;Transwell实验显示,IL-22作用下,侵袭过膜细胞测得OD值较空白对照组增加,其中CAPAN-2细胞刺激前后增长率大于SW1990细胞;RT-PCR结果显示,IL-22可以显著增强VEGF、HMGB1、MMP9的mRNA表达水平,其中CAPAN-2细胞受IL-22刺激后三种促癌因子mRNA表达增强率均大于SW1990细胞;Western blot结果显示,IL-22可以明显增强STAT3通路磷酸化,STAT3蛋白表达下降,p-STAT3蛋白表达增加,其下游VEGF、HMGB1、MMP9的蛋白表达水平显著上升,其中CAPAN-2细胞的变化率大于SW1990细胞的变化率,以上实验结果差异均有统计学意义(P<0.05)。结论:IL-22通过磷酸化STAT3通路上调肿瘤增殖、迁移和侵袭相关因子VEGF、HMGB1、MMP9的mRNA及蛋白表达,从而促进胰腺癌细胞增殖、迁移和侵袭,并且随着胰腺癌恶性程度的增加,其促增殖、迁移和侵袭效应增强。

Objective:To investigate the effect of interleukin-22(IL-22)on the proliferation,migration and invasion of human pancreatic cancer cells and its mechanism.To explore the effect of IL-22 on pancreatic cancer cell lines with different differentiation degree.Methods:SW1990 and CAPAN-2 were choose as two kinds of pancreatic ductal carcinoma cells to do parallel experiments according to the degree of differentiation.MTT assay was used to detect the effect of IL-22 on the proliferation of two kinds of pancreatic cancer cells.Cell scratch assay was used to observe the effect of IL-22 on the migration ability of two pancreatic cancer cell lines.Transwell invasion assay was used to detectthe effect of IL-22 on the invasion ability.Detection of RT-PCR before and after the IL-22 treatment in cells was done,and it was about the difference of the VEGF(vascular endothelial growth factor),HMGB1(high mobility group protein B1),MMP9(matrix metalloproteinase 9)mRNA expression.Western blot assay before and after the IL-22 treatment,its about the difference of STAT3,p-STAT3,VEGF,HMGB1,MMP9 protein expression in the cells and parallel comparisons between the two pancreatic cancer cell lines were done above all the experiments.Results:In the MTT experiment,under the effects of IL-22 on the different concentration,OD values measured were the same with increasing concentration increased gradually,CAPAN-2 cell growth rate is greater than the growth rate of SW1990 cells.Scratch test results after 24 h and 48 h showed that the relative scratch width decreases by IL-22.The 24 h and 48 h change rate of CAPAN-2 cells was greater than the rate of SW1990 cells.Transwell results showed that,stimulated by IL-22,the OD value increased compared with blank control group,the growth rate of CAPAN-2 cells was greater than that of SW1990 cells.RT-PCR results showed that after IL-22 stimulation can significantly enhance the mRNA expression level of VEGF,HMGB1,MMP9,three kinds of cancer promoting factor mRNA expression increased more in CAPAN-2 than in SW1990.Western blot results showed that IL-22 can significantly enhance the phosphorylation of STAT3 pathway,STAT3 protein expression decreased,p-STAT3 protein expression increased,and the downstream factor VEGF,HMGB1 and MMP9 protein expression increased significantly,the change rate of CAPAN-2 cells is greater than the change rate of SW1990 cells,there was a very significant difference between both groups among experiments above(P<0.05).Conclusion:IL-22 can promote the proliferation and metastasis of pancreatic cancer cells by phosphorylated STAT3 pathway to raise the expression of RNA and protein of tumor metastasis and invasion related factors as VEGF,HMGB1 and MMP9,at the same time,with the increase of malignant degree of pancreatic cancer,the effect of proliferation and metastasis was enhanced.