miR-491-5p靶向FGFR4调节GSK3β/β-catenin信号通路对小儿肝母细胞瘤增殖和迁移的影响

Effects of MiR-491-5p/FGFR4-Regulated GSK3β/β-catenin Signaling Pathway on the Proliferation and Migrationof Pediatric Hepatoblastoma

目的探讨miR-491-5p靶向FGFR4调节GSK3β/β-catenin信号通路对小儿肝母细胞瘤增殖和迁移的影响。方法qRT-PCR检测小儿肝母细胞瘤组织、瘤旁正常组织、正常肝细胞LO2和肝母细胞瘤细胞HuH-6中miR-491-5p和FGFR4 mRNA的表达,Western blot检测FGFR4蛋白表达;荧光素酶活性分析实验检测miR-491-5p与FGFR4的靶向关系;mimic NC,miR-491-5p mimic、pcDNA3.1质粒和pcDNA3.1-FGFR4质粒分别转染HuH-6细胞,qRT-PCR与Western blot检测FGFR4的表达;CCK-8实验检测HuH-6细胞增殖能力的变化,细胞迁移实验分析HuH-6细胞迁移能力的变化;Western blot实验检测HuH-6细胞中GSK3β/β-catenin信号通路相关蛋白p-GSK3β、p-β-catenin和p-C-myc蛋白的表达。结果小儿肝母细胞瘤组织和肝母细胞瘤细胞HuH-6中miR-491-5p表达量降低,FGFR4表达量升高;荧光素酶活性分析实验表明miR-491-5p靶向调控FGFR4;qRT-PCR和Western blot实验表明miR-491-5p抑制FGFR4表达;CCK-8和细胞迁移实验表明,miR-491-5p mimic抑制肝母细胞瘤细胞HuH-6的增殖和迁移,而pcDNA3.1-FGFR4可逆转miR-491-5p mimic对HuH-6细胞增殖和迁移的抑制作用;Western blot实验显示,miR-491-5p mimic可抑制p-GSK3β、p-β-catenin和p-C-myc蛋白的表达,pcDNA3.1-FGFR4可逆转miR-491-5p mimic对p-GSK3β、p-β-catenin和p-C-myc蛋白的抑制作用。结论miR-491-5p负向调节FGFR4,抑制肝母细胞瘤细胞的增殖和迁移,可能与GSK3β/β-catenin信号通路失活相关。

Objective To investigate the effect of miR-491-5p/FGFR4-regulated GSK3β/β-catenin signaling pathway on the proliferation and migration of pediatric hepatoblastoma.Methods The expressions of miR-491-5p and FGFR4 mRNA were detected by qRT-PCR,and the protein level of FGFR4 was detected by western blot in adjacent normal tissues,pediatric hepatoblastoma tissues,normal hepatocytes cell line LO2 and hepatoblastoma cell line HuH-6.The relationship between miR-491-5 and FGFR4 was detected by a dual luciferase assay.The miR-491-5p mimic,mimic NC,pcDNA3.1 plasmid and pcDNA3.1-FGFR4 plasmid were transfected into HuHu-6 cells to detect the expression of FGFR4 by qRT-PCR and western blot,while proliferation and invasion of cells were measured by CCK-8 and transwell assay,respectively.The expressions of p-GSK3β,p-β-catenin and p-C-myc in GSK3β/β-catenin signaling pathway were detected by western blot.Results In pediatric hepatoblastoma tissues and hepatoblastoma cells HuH-6,the expression of miR-491-5 was down-regulated,while the expression of FGFR4 was up-regulated.Luciferase activity analysis revealed that miR-491-5p targeted FGFR4.qRT-PCR and western blot showed that miR-491-5p inhibited FGFR4.CCK-8 and transwell assays indicated that miR-491-5p mimic inhibited proliferation and migration of HuH-6 cells,while pcDNA3.1-FGFR4 reversed the inhibitory effect of miR-491-5p mimic on proliferation and migration of HuH-6 cells.Western blot experiments showed that miR-491-5p mimic could inhibit the protein levels of p-GSK3β,p-β-catenin and p-C-myc,while pcDNA3.1-FGFR4 reversed the inhibitory effect of miR-491-5p mimic on the protein levels of p-GSK3β,p-β-catenin and p-C-myc.Conclusion miR-491-5p can negatively regulate FGFR4 to suppress proliferation and migration of hepatoblastoma cells,which might involve the inactivation of GSK3β/β-catenin signaling pathway.