微小RNA-100靶向成纤维生长因子受体调控胰腺癌细胞的增殖

摘要

目的 探讨微小RNA(miR)-100对成纤维生长因子受体3(FGFR3)调控及其对胰腺癌细胞增殖的影响.方法 采用实时PCR检测17对胰腺癌和癌旁组织中miR-100的表达水平.应用CCK-8试剂盒检测过表达miR-100对胰腺癌细胞株增殖的影响.利用分子生物学技术构建FGFR3的野生型及突变型3’-UTR报告基因,分析miR-100对FGFR3的调控作用,然后采用实时PCR和蛋白印迹检测过表达miR-100对FGFR3 mRNA和蛋白表达水平的影响.应用CCK-8、5-溴脱氧尿嘧啶核苷(Edu)探讨特异性敲低FGFR3对胰腺癌细胞株增殖的影响.结果 miR-100在胰腺癌组织中呈低表达水平(P＜0.05).过表达miR-100可显著抑制胰腺癌细胞的增殖(P＜0.05).报告基因分析显示miR-100能够显著下调FGFR3的野生型3’-UTR报告基因的荧光表达(P＜0.05),而对突变型报告基因和空白对照组的荧光表达影响不大.实时PCR和蛋白印迹检测显示miR-100可显著抑制FGFR3的mRNA和蛋白表达(P＜0.05).特异性敲低FGFR3后明显抑制胰腺癌细胞的增殖(P＜0.05).结论 miR-100可通过调控FGFR3表达来抑制胰腺癌细胞的增殖,从而在胰腺癌中发挥抑癌基因的功能.%Objective To investigate the effects of miR-100 on the proliferation of MIA PaCa-2 and CFPAC-1 cells through targeting fibroblast growth factor receptor 3 (FGFR3).Methods miR-100 expression levels in 17 cancer tissues and 17 nonmalignant tissues were examined by Real-time PCR.The effect of miR-100 overexpression on cell proliferation was examined by CCK-8 assay in vitro.Luciferase assay was used to confirm that miR-100 could directly target FGFR3.Real-time PCR and Western blot were used to examine the expression of FGFR3 in miR-100 overexpressing pancreatic cancer cells.The predicted target gene of miR-100,FGFR3,was downregulated by siRNA,and its effect on cell proliferation was also examined.Cell proliferation was analyzed using CCK-8 and Edu assay.Results miR-100 was lowly expressed in pancreatic cancer tissues (P ＜ 0.05).In pancreatic cancer cells,the transfection of lv-miR-100 was able to upregulate the endogenous expression of miR-100 and inhibit the cell proliferation (P ＜0.05).Luciferase assay showed FGFR3 was the direct target of miR-1O0.FGFR3 was significantly downregulated by overexpressing miR-100 in pancreatic cancer cells (P ＜0.05),and FGFR3 knockdown by specific siRNA exerted the similar effect as miR-100 overexpression (P ＜ 0.05).Conclusions Our study identified a new miRNA regulator,miR-100,and clarified a novel mechanism of how miR-100 regulates cell proliferation in pancreatic cancer.The strategy of overexpressing the tumor suppressor miR-100 may provide a new therapeutic approach for treating patients with pancreatic cancer.