miR-328-3p靶向下调FOXP3抑制IL-17诱导的Caco-2人结直肠癌细胞侵袭和迁移

miR-328-3p down-regulates FOXP3 to inhibit IL-17-induced invasion and migration of Caco-2 human colorectal cancer cells

目的研究miR⁃328⁃3p对白细胞介素17(IL⁃17)诱导的结直肠癌Caco⁃2细胞转移的影响和机制。方法实时定量PCR检测Caco⁃2结直肠上皮细胞和结直肠癌细胞中miR⁃328⁃3p、叉头盒P3(FOXP3)和IL⁃17 mRNA表达。将miR⁃328⁃3p模拟物(mimics)转染至结直肠癌Caco⁃2细胞中,然后给予IL⁃17诱导处理后,Transwell^(TM)小室测定细胞侵袭和迁移,明胶酶谱法测定细胞中基质金属蛋白酶2(MMP2)和MMP9活性。双荧光素酶报告基因实验验证miR⁃328⁃3p与FOXP3的靶向调控关系,蛋白质印迹法检测miR⁃328⁃3p mimics对Caco⁃2细胞中FOXP3蛋白表达的影响。将FOXP3过表达载体(pcDNA3.1⁃FOXP3)和miR⁃328⁃3p mimics共转染到Caco⁃2细胞,利用上述相同方法测定细胞侵袭、迁移和细胞MMP2、MMP9活性。结果与结直肠上皮细胞比较,miR⁃328⁃3p在结直肠癌细胞中的表达水平降低,FOXP3和IL⁃17 mRNA水平升高。转染miR⁃328⁃3p mimics可降低IL⁃17诱导处理后的Caco⁃2细胞侵袭和迁移能力及细胞MMP2、MMP9活性。miR⁃328⁃3p靶向结合并抑制Caco⁃2细胞FOXP3蛋白表达。转染pcDNA3.1⁃FOXP3可降低miR⁃328⁃3p mimics对IL⁃17诱导的Caco⁃2细胞侵袭、迁移及细胞MMP2、MMP9活性的影响。结论miR⁃328⁃3p可能通过靶向抑制FOXP3减弱IL⁃17诱导的结直肠癌Caco⁃2细胞的侵袭和迁移能力。

Objective To investigate the effect of miR⁃328⁃3p on the invasion and migration of colorectal cancer cells induced by interleukin⁃17(IL⁃17)and its mechanism.Methods Real⁃time quantitative PCR was used to detect the expressions of miR⁃328⁃3p,FOXP3,and IL⁃17 mRNA in FHC colorectal epithelial cells and colorectal cancer cells(HCT116,Caco⁃2,and LoVo).Transwell^(TM) assay was used to detect cell invasion and migration,and gelatin zymography assay to detect the activities of matrix metalloproteinases 2(MMP2)and MMP9.Dual luciferase reporter assay was conducted to detect the targeted regulation relationship between miR⁃328⁃3p and FOXP3.The level of FOXP3 protein in Caco⁃2 cells was detected by Western blot analysis.Results Compared with those in colorectal epithelial cells,the expression level of miR⁃328⁃3p in colorectal cancer cells decreased,while the levels of FOXP3 and IL⁃17 increased.Transfection of miR⁃328⁃3p mimics inhibited the invasion and migration ability of IL⁃17⁃induced Caco⁃2 cells and the activities of MMP2 and MMP9 in the cells.miR⁃328⁃3p directly targeted and inhibited the protein level of FOXP3 in Caco⁃2 cells.Overexpression of FOXP3 partly reversed the effect of miR⁃328⁃3p mimics on the invasion and migration ability of IL⁃17⁃induced Caco⁃2 cells and the activities of MMP2 and MMP9 in the cells.Conclusion miR⁃328⁃3p inhibits the invasion and migration of IL⁃17⁃induced Caco⁃2 cells by down⁃regulating FOXP3.