miR-101通过靶向FGF2抑制非小细胞肺癌的迁移和侵袭  

miR-101 inhibits migration and invasion of non-small cell lung cancer by targeting fibroblast growth factor 2

作　　者：郭雪茹 张其程[1] 曹丽敏 徐克[1] GUO Xueru;ZHANG Qicheng;CAO Limin;XU Ke(Tianjin Key Laboratory of Lung Cancer Metastasis and Tumor Microenviron‐ment,Tianjin Lung Cancer Institute,Tianjin Medical University General Hospital,Tianjin 300052,China)

机构地区：[1]天津医科大学总医院,天津市肺癌研究所,天津市肺癌转移与肿瘤微环境重点实验室,天津300052

出　　处：《中国肿瘤生物治疗杂志》2020年第9期984-991,共8页Chinese Journal of Cancer Biotherapy

基　　金：国家自然科学基金资助项目(No.81372519);教育部博士点基金资助项目(No.20131202110005);天津市自然科学基金重点资助项目(No.18JCZDJC98500)。

摘　　要：目的:探究microRNA-101(miR-101)通过靶向成纤维细胞生长因子2(fibroblast growth factor 2,FGF2)抑制非小细胞肺癌(non-small cell lung cancer,NSCLC)细胞迁移和侵袭的分子机制。方法:采用qPCR法检测人正常肺上皮细胞BEAS-2B和NSCLC细胞系A549、H661和SK-MES-1,以及转染后A549细胞miR-101和FGF2的表达水平。分别将miR-NC、miR-101 mimics、miR-IN-NC、miR-101 inhibitor或pcDNA-3.1空质粒、pcDNA-FGF2转染至A549细胞,运用划痕愈合实验和Transwell小室实验,检测A549细胞中过表达miR-101和FGF2对NSCLC细胞迁移和侵袭能力的影响,采用Western blotting(WB)法检测各组A549细胞中FGF2、E-cadherin、N-cadherin、Vimentin、ERK1/2和p-ERK1/2的表达水平。结果:miR-101在NSCLC细胞系中的表达水平明显低于正常肺上皮细胞(均P<0.05),而以A549细胞中表达水平为最低。过表达miR-101可明显抑制A549细胞的迁移(P<0.05)和侵袭(P<0.01),且使细胞中E-cadherin的表达增多(P<0.05)而Vimentin(P<0.05)、N-cadherin(P<0.01)和p-ERK1/2(P<0.05)的表达水平降低。抑制miR-101表达后,可以显著增强A549细胞的迁移和侵袭能力(均P<0.05),引起细胞中E-cadherin表达明显降低而Vimentin、N-cadherin和p-ERK1/2表达水平增高(均P<0.05)。采用WB法和双荧光素酶报告基因实验验证了FGF2是miR-101的直接靶基因,且过表达FGF2后显著增强A549细胞的迁移和侵袭能力(均P<0.01),以及减少细胞中E-cadherin的表达(P<0.01)而增加Vimentin(P<0.01)、N-cadherin(P<0.05)和p-ERK1/2(P<0.05)表达水平。与单独过表达FGF2组相比,共同过表达miR-101和FGF2组A549细胞迁移和侵袭能力明显减弱(均P<0.01),其E-cadherin的表达增多(P<0.01)而Vimentin(P<0.01)、N-cadherin(P<0.05)和p-ERK1/2表达水平下降(P<0.01)。结论:miR-101通过调控靶基因FGF2抑制NSCLC A549细胞的上皮间质转化(EMT)过程及ERK信号通路,进而抑制NSCLC细胞的迁移和侵袭。Objective:To investigate the molecular mechanism of microRNA-101(miR-101)inhibiting the migration and invasion of non-small cell lung cancer(NSCLC)via targeting fibroblast growth factor 2(FGF2).Methods:qPCR was used to detect the expression levels of miR-101 and FGF2 in human normal lung epithelial BEAS-2B cells and NSCLC cell lines(A549,H661 and SK-MES-1)as well as A549 cells after transfection.MiR-NC,miR-101 mimics,miR-IN-NC,miR-101 inhibitor or pcDNA-3.1 empty plasmid,pcDNA-FGF2 were respectively transfected into A549 cells.Wound healing assay and Transwell assay were used to examine the effects of overexpression of miR-101 and FGF2 on the migration and invasion of A549 cells.Western blotting(WB)was used to detect the expression levels of FGF2,E-cadherin,N-cadherin,Vimentin,ERK1/2 and p-ERK1/2 in A549 cells in each group.Results:The expression level of miR-101 in NSCLC cell lines were significantly lower than that in normal lung epithelial cells(all P<0.05),while the expression level in A549 cells was the lowest.Overexpression of miR-101 significantly inhibited the migration(P<0.05)and invasion(P<0.01)of A549 cells,increased the expression level of E-cadherin but decreased the expression level of Vimentin(P<0.05),N-cadherin(P<0.01)and p-ERK1/2(P<0.05).Inhibition of miR-101 significantly enhanced the invasion and migration of A549 cells(all P<0.05),decreased the expression level of E-cadherin but increased the expression levels of Vimentin,N-cadherin and p-ERK1/2(all P<0.05).The results of WB and Dual-luciferase reporter gene assay verified that FGF2 is a direct target gene of miR-101,and over-expression of FGF2 significantly enhanced the invasion and migration of A549 cells(all P<0.01),decreased the expression of E-cad-herin(P<0.01)but increased the expressions of Vimentin(P<0.01),N-cadherin(P<0.05)and p-ERK1/2(P<0.01).Compared with the FGF2 overexpression alone group,co-overexpression of miR-101 and FGF2 significantly reduced the invasion and migration of A549 cells(all P<0.01),increased the expression of E-c

关 键 词：miR-101 非小细胞肺癌 A549细胞 迁移 侵袭 成纤维细胞生长因子2 上皮间质转化 ERK信号通路 

分 类 号：R734.2[医药卫生—肿瘤] R730.5[医药卫生—临床医学]