摘要
目的:研究微RNA-490-5p(microRNA-490-5p,miR-490-5p)在人肝内胆管细胞癌组织中的表达情况,并探讨其对肝内胆管癌RBE细胞侵袭和迁移能力的影响及可能的分子机制。方法:采用实时荧光定量PCR法检测miR-490-5p在5对肝内胆管细胞癌组织及其癌旁正常组织中的表达水平。利用脂质体瞬时转染法将miR-490-5p模拟物及其阴性对照转入人肝内胆管细胞癌RBE细胞中。随后,采用划痕愈合实验以及Transwell小室迁移实验检测RBE细胞迁移能力的变化,Transwell小室侵袭实验检测RBE细胞侵袭能力的变化;最后,通过CCK-8法、平板克隆形成实验和FCM法检测RBE细胞增殖及凋亡的变化。应用在线miRNA靶基因预测软件预测miR-490-5p的靶基因,并采用蛋白印迹法对预测的靶基因进行验证。结果:miR-490-5p在人肝内胆管细胞癌组织中明显下调(P<0.05)。与阴性对照组相比,miR-490-5p模拟物转染组RBE细胞的增殖、克隆形成能力和凋亡均无明显变化(P值均>0.05);划痕愈合实验以及Transwell小室迁移实验检测结果显示,RBE细胞的迁移能力被明显抑制(P值均<0.05);Transwell小室侵袭实验检测结果显示,RBE细胞的侵袭能力被明显抑制(P值<0.05)。Target Scan网站预测转录因子FBJ骨肉瘤癌基因(FBJ osteosarcoma oncogene,c-Fos)可能是miR-490-5p的潜在靶基因;蛋白质印迹法证实,miR-490-5p模拟物转染组细胞中c-Fos蛋白的表达水平明显下调(P<0.05)。结论:miR-490-5p在人肝内胆管细胞癌组织中低表达,而且其可能通过下调c-Fos的表达来调控人肝内胆管细胞癌RBE细胞的迁移和侵袭。
Objective： To investigate the expression of microRNA-490-5p （miR490-5p） in human intrahepatic cholangiocarcinoma tissues,and to study its impacts on invasion and migration of intrahepatic cholangiocarcinoma RBE cells and the underlying molecular mechanism.
Methods： The expression level of miR-490-5p in 5 pairs of intrahepatic cholangiocarcinoma tissues and the adjacent normal tissues was detected by real-time fluorescent quantitative PCR.Intrahepatic cholangiocarcinoma RBE cells were transfected with miR-490-5p-mimics and the negative control by LipofectAMINE 2000,respectively.Then the cell migration and invasion abilities were explored by scratch wound healing assay and Transwell chamber assay,the proliferation of RBE cells were measured by CCK-8 assay,the colony formation ability was detected by colony formation assay,the apoptosis rate was detected by FCM method.The target genes of miR-490-5p were predicted by online miRNA-targeted gene prediction software,and validated by Western blotting.
Results： The expression level of miR-490-5p was significantly down-regulated in human intrahepatic cholangiocarcinoma tissue as compared with the adjacent normal tissues （P 〈 0.05）.After transfection with miR-490-5p-mimics,the proliferation,colony forming and apoptosis of RBE cells were not significantly changed （all P 〉 0.05）,but the scratch healing,migration and invasion abilities of RBE cells were significantly decreased （all P 〈 0.05）.TargetScan website predicted that the potential target gene of miR-490-5p was FBJ osteosarcoma oncogene （c-Fos）,and Western blotting verified that the expression of c-Fos was significantly down-regulated in RBE cells transfected with miR-490-5p-mimics.
Conclusion： miR-490-5p is lowly expressed in human intrahepatic cholangiocarcinoma,and may regulate the migration and invasion of intrahepatic cholangiocarcinoma RBE cells by down-regulating the expression of c-Fos gene.
出处
《肿瘤》
CAS
CSCD
北大核心
2017年第7期700-709,共10页
Tumor
基金
国家自然科学基金资助项目(编号:81272402)~~
