微小RNA507靶向调控VEGFC表达及对肝癌细胞生物学行为和PI3K／Akt通路的影响#br#

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摘要/Abstract

摘要： 目的探讨微小RNA507（miR507）对肝癌细胞增殖、凋亡和迁移能力及PI3K／Akt信号通路的影响并分析miR507对血管内皮生长因子C（VEGFC）的靶向调控作用。
方法采用实时荧光定量PCR（QPCR）检测2014年1月至2017年3月经病理确诊的90例肝癌患者的癌组织及癌旁组织中的miR507和VEGFC mRNA水平；采用脂质体法向人肝癌细胞HepG2转染miR507 模拟物（过表达组）或阴性对照（NC组），QPCR检测转染48 h后的miR507水平，MTT法比较不同处理时间（0、12、24、48 h）各组细胞的增殖情况，Annexin ⅤFITC／PI流式细胞术检测细胞凋亡情况，划痕实验比较各组迁移情况，QPCR和Western blotting分别检测VEGFC及PI3K／Akt信号通路中pPI3K和pAkt的mRNA和蛋白水平；采用双荧光素酶报告基因实验验证miR507与靶基因VEGFC间的靶向关系和结合位点。
结果QPCR检测发现肝癌组织中的miR507水平为0672±0089，低于癌旁组织的1142±0136，VEGFC mRNA水平为1482±0156，高于癌旁组织的1021±0087，差异均有统计学意义（P＜005）。与NC组和对照组相比，过表达组HepG2细胞的增殖活力在转染24～48 h后明显减弱（P＜001）。过表达组的凋亡率为（1726±196）％，高于NC组的（785±087）％和对照组的（813±069）％，过表达组的愈合率为（3965±487）％，低于NC组的（5818±273）％和对照组的（5724±317）％，差异有统计学意义（P＜005）。过表达组的pAkt、pPI3K和VEGFC的蛋白和mRNA水平均低于NC组和对照组，差异有统计学意义（P＜005）。双荧光素酶报告基因实验证明VEGFC是miR507的直接作用靶点。
结论VEGFC可能通过PI3K／Akt信号通路介导miR507对肝癌细胞HepG2增殖、凋亡与迁移能力的调控，因此miR507可作为肝癌潜在的分子治疗靶点。

关键词: 肝癌, 微小RNA507, 增殖, 凋亡, 迁移, 血管内皮生长因子C

Abstract: ObjectiveTo investigate the effect of microRNA507 （miR507） on the proliferation， apoptosis， migration and PI3K／Akt signaling pathway of hepatoma cells and analyze the targeting regulation of miR507 on vascular endothelial growth factor C （VEGFC）.
MethodsQuantitative realtime polymerase chain reaction （QPCR） was used to detect the levels of miR507 and VEGFC mRNA in the cancer tissues and paracancerous tissues of 90 patients with hepatoma cancer from January 2014 to March 2017. Human hepatoma cells HepG2 were transfected with miR507 mimics （overexpressing group） or negative control （NC group） by liposome method. QPCR was used to detect the miR507 level after 48 htransfection. MTT method was used to compare the cell proliferation at different treatment times （0， 12， 24， 48 h）. Flow cytometry with Annexin ⅤFITC／PI staining was used to detect the cell apoptosis， and the scratch test was used to compare the migration of each group. QPCR and Western blotting were employed to detect the mRNA and protein levels of VEGFC and PI3K／Akt signal pathwaysrelated genes including pAkt and pPI3K. Dual luciferase reporter gene test was used to verify the target relationship and binding site between miR507 and target gene VEGFC.
ResultsQPCR detection showed that the level of miR507 in hepatoma cancer tissues was 0672±0089， lower than 1142±0136 in paracancerous tissues and level of VEGFC mRNA in hepatoma cancer tissues was 1482±0156， higher than 1021±0087 in paracancerous tissues （P＜005）. Compared with the NC group and the control group， the proliferative activity of HepG2 cells in the overexpressing group was significantly decreased after transfection of 2448 h （P＜001）. The apoptotic rate of HepG2 cells in overexpressing group was （1726±196）％， higher than （785±087）％ of NC group and （813±069）％ of control group （P＜005）. The healing rate of HepG2 cells in overexpressing group was （3965±487）％， lower than （5818±273）％ in NC group and （5724±317）％ in control group （P＜005）. The protein and mRNA levels of pAkt， pPI3K and VEGFC in the overexpression group were lower than those in the NC group and the control group （P＜005）. The double luciferase reporter gene experiment proved that VEGFC was a direct target of miR507.
ConclusionMiR507 may regulate HepG2 cells proliferation， apoptosis and migration through targeting VEGFC of PI3K／Akt signal pathway， and serve as a potential therapeutic strategy in hepatoma cancer.

Key words: Hepatoma cancer, MicroRNA507, Proliferation, Apoptosis, Migration, Vascular endothelial growth factor C

牛虹, 周浩本, 刘怀民, 高启龙, 田同德, 马东阳, 唐静雯. 微小RNA507靶向调控VEGFC表达及对肝癌细胞生物学行为和PI3K／Akt通路的影响#br#

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[J]. 临床肿瘤学杂志, 2018, 23(8): 673-679.

NIU Hong, ZHOU Haoben, LIU Huaimin, GAO Qilong, TIAN Tongde, MA Dongyang, TANG Jingwen. . Regulation of VEGFC expression by microRNA507 and its effect on biological behavior and PI3K／Akt pathway of hepatoma cells[J]. Chinese Clinical Oncology, 2018, 23(8): 673-679.

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