摘要
目的探讨微RNA（miR）-758对肝癌细胞转移的影响及其作用机制，研究miR-758对促癌基因星形细胞上调基因1（AEG-1）的调控作用。方法用miR-758瞬时转染HepG2细胞，通过Transwell迁移和侵袭实验，观察miR-758对肿瘤细胞转移的影响。利用CCK8法检测细胞增殖活性，观察miR-758对肿瘤细胞增殖的影响。使用流式细胞仪定量分析细胞周期，观察miR-758对细胞周期的影响。通过上皮间充质转化（EMT）实验，观察miR-758是否影响肝癌细胞EMT。用miR-758瞬时转染人脐静脉内皮细胞（HUVEC），观察miR-758对管腔形成的影响。软件预测显示miR-758的靶基因为AEG-1。将含miR-758结合位点的AEG-1 3′UTR构建到荧光素酶表达载体中，用miR-758和AEG-1基因3′UTR载体共同转染HepG2细胞，验证miR-758的靶基因是否为AEG-1。用miR-758瞬时转染HepG2细胞，检测AEG-1蛋白的表达量变化。结果miR-758过表达可抑制HepG2细胞的迁移、侵袭，抑制细胞增殖和周期，抑制HepG2细胞EMT，抑制HUVEC细胞管腔形成。miR-758和AEG-1基因3′UTR质粒共同转染HepG2细胞后，荧光素酶的表达量降低。miR-758靶点AEG-1 3′UTR突变后，荧光素酶的表达量恢复。另外，细胞中miR-758含量升高时，Western Blot检测到AEG-1蛋白水平的表达量降低。结论miR-758负调控癌症转移的多个步骤，包括细胞迁移、侵袭、增殖、EMT以及血管新生等。miR-758下游靶基因为AEG-1。
Objective To investigate the mechanism of miR-758 in hepatocellular carcinoma cell HepG2, and to investigate the regulatory role of miR-758 on astrocyte elevated gene-1 （AEG-1）. Methods Transient transfection of miR-758 into HepG2 cells was performed to study the effect of miR-758 on tumor cell metastasis by transwell migration and invasion experiments. CCK8 assay was used to detect the cell proliferation activity. The cell cycle was analyzed by flow cytometry. The effect of miR-758 on epidermal mesenchymal transition （EMT） was determined by the expression of EMT markers. Transient transfection of miR- 758 into human umbilical vein endothelial cells （HUVECs） was performed to explore the effect of miR-758 on luminal formation. AEG-1 3′UTR containing the binding site of miR-758 was constructed into luciferase expression vector. The miR-758 and the vector was co-transfected into HepG2 cells. And then the change in expression level of AEG-1 protein was detected through Western Blot. Results The overexpression of miR- 758 inhibited HepG2 cell migration and invasion, as well as the cell proliferation and the cell cycle. The miR-758 was also found to inhibit EMT of HepG2 cells and the lumen formation of HUVEC cells. After the co-transfection of miR-758 with the plasmid containing AEG-1 gene 3′UTR into HepG2 ceils, the luciferase expression was decreased. The luciferase expression was restored when the binding site of miR-758 in the 3′ UTR was mutated. Further evidence by Western Blot showed the protein level of AEG-1 in HepG2 cells was significantly decreased after transfection of miR-758. Conclusions The miR-758 negatively regulates multiple steps during cancer metastasis, including cell migration, invasion, cell proliferation, EMT, as well as angiogenesis. And AEG-1 has been identified as a downstream target of miR-758.
出处
《中华肝胆外科杂志》
CSCD
北大核心
2017年第10期689-693,共5页
Chinese Journal of Hepatobiliary Surgery
基金
北京市自然科学基金（7174314）
国家自然科学基金面上项目（81472328）
国家科技支撑计划（2015BAI02B00）
首都临床特色应用研究与成果推广（Z171100001017063）
