华中科技大学同济医学院附属武汉中心医院 普通外科，湖北 武汉 430014
何涛， Email: firstname.lastname@example.org
Department of General Surgery, the Central Hospital of Wuhan, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430014, China
背景与目的 miR-200a-3p参与了多种肿瘤的生物学过程的调控，但在不同肿瘤中起着不同的作用（促癌基因或抑癌基因）。目前，其在胆囊癌中的作用尚不清楚。因此，本研究探讨miR-200a-3p在胆囊癌中的表达及其对胆囊癌细胞生物学功能的影响与机制。方法 采用qRT-PCR法测定32例胆囊癌及癌旁组织、胆囊癌细胞系（GBC-SD、SGC-996、NOZ）及正常人胆囊上皮细胞系HGBEC中miR-200a-3p的表达。采用Lipofectamine? 3000试剂盒将GBC-SD及NOZ细胞系分别转染miR-200a-3p模拟物（miR-200a-3p过表达组）、miR-200a-3p抑制物序列（miR-200a-3p沉默组）及阴性对照序列（阴性对照组）。MTT实验测定细胞增殖能力，Transwell实验测定细胞侵袭能力，MiRBD/Targetscan7.2/starBase2.0/miRtarbase网站预测miR-200a-3p的下游靶基因，并采用荧光素酶实验验证。Western blot检测上述3组细胞中靶基因与上皮间质转化（EMT）相关分子蛋白（E-cadherin、vimentin）的表达。结果 qRT-PCR结果显示，胆囊癌组织中miR-200a-3p表达量低于癌旁组织，所有胆囊癌细胞系中miR-200a-3p表达量低于正常人胆囊上皮系HGBEC（均P<0.05）。GBC-SD及NOZ细胞系转染后，与各自的阴性对照组比较，两种细胞的miR-200a-3p过表达组miR-200a-3p表达量明显升高、增殖与侵袭能力明显减弱，两种细胞的miR-200a-3p沉默组miR-200a-3p表达量明显降低、增殖与侵袭能力明显增强（均P<0.05）。在线网站预测显示，miR-200a-3p和Notch2存在潜在结合位点；荧光素酶实验显示，miR-200a-3p导致Notch2野生型质粒pmirGLO-Notch2-3'UT WT荧光素酶活性明显降低，而miR-200a-3p对Notch2突变型质粒pmirGLO-Notch2-3'UTR MUT的荧光素酶活性没有影响。Western blot结果显示，两种细胞的miR-200a-3p过表达组与各自的阴性对照组比较，E-cadherin蛋白表达量升高、vimentin蛋白与Notch2蛋白表达量降低，而3种蛋白在两种细胞的miR-200a-3p沉默组则呈相反的变化（均P<0.05）
Background and Aims MiR-200a-3p participates in the regulation of biological processes in a variety of tumors, but it exerts different effects in different tumors (oncogene or tumor suppressor). At present, its role in gallbladder cancer is still unclear. Therefore, this study was conducted to investigate the expression of miR-200a-3p in gallbladder cancer as well as its effects on biological behaviors of gallbladder cancer cells and the mechanism.Methods The expressions of miR-200a-3p in 32 specimens of gallbladder cancer tissue and adjacent tissue as well as in different gallbladder cancer cell lines (GBC-SD, SGC-996, and NOZ) and normal human gallbladder epithelial cell line HGBEC were determined by qRT-PCR method. The GBC-SD and NOZ cell lines were transfected with miR-200a-3p-mimics (miR-200a-3p overexpression group), miR-200a-3p-inhibitors (miR-200a-3p silencing group) and scramble sequences (negative control group) respectively using Lipofectamine? 3000 kit. The proliferation and invasion abilities of the cells were examined by MTT assay and Transwell assay. The downstream target genes of miR-200a-3p were predicted using MiRBD/Targetscan7.2/starBase2.0/miRtarbase website, and were verified by Luciferase experiment. The protein expressions of the target genes and the molecules (E-cadherin and vimentin) associated with epithelial-to-mesenchymal transition (EMT) in above three groups of cells were measured by Western blot analysis.Results The results of qRT-PCR showed that the expression level of miR-200a-3p in gallbladder cancer tissue was lower than that in adjacent tissue, and the expression levels of miR-200a-3p in all gallbladder cancer cell lines were lower than that in normal human gallbladder epithelial cell line HGBEC (all P<0.05). In GBC-SD and NOZ cell lines after transfection, the expressions of miR-200a-3p were significantly increased and the proliferative and invasion abilities were significantly decreased in miR-200a-3p overexpression groups of both cell lines, while the expressions of miR-200a-3p were significantly decreased and the proliferative and invasion abilities were significantly increased in miR-200a-3p silencing groups of both cell lines compared with their respective negative control groups (all P<0.05). Online website prediction showed that there were potential binding sites between miR-200a-3p and Notch2. Luciferase verification experiments showed that miR-200a-3p caused markedly decreased luciferase activity in Notch2 wild-type plasmid pmirGLO-Notch2-3'UT WT, but exerted no effect on the luciferase activity of Notch2 mutant plasmid pmirGLO-Notch2-3'UTR MUT. The results of Western blot analysis showed the E-cadherin protein expressions were increased and the protein expressions of vimentin and Notch2 were decreased in miR-200a-3p overexpression groups of both cell lines, while the opposite changes in the three proteins were observed in miR-200a-3p silencing groups of both cell lines compared with their respective negative control groups (all P<0.05).Conclusion The expression of miR-200a-3p is decreased in gallbladder cancer, and it may play a tumor suppressor role in gallbladder cancer, and the mechanism may be probably associated with its down-regulating Notch2 and thereby inhibiting the EMT process.