微小RNA320靶向FOXM1调控食管鳞癌放射敏感性的机制研究#br#

摘要/Abstract

摘要： 目的探讨微小RNA320（miR320）对食管鳞癌（ESCC）放射敏感性的影响及可能机制。
方法采用实时荧光定量PCR（QPCR）检测正常食管上皮细胞株HEEC和ESCC细胞株KYSE150、TE13和 Eca109中miR320和叉头框蛋白M1（FOXM1）的表达水平。采用脂质体法将miR320 模拟物（mimics）及其阴性对照（NC）转染至Eca109细胞，分为转染组和对照组。QPCR和Western blotting检测miR320 mimics转染后FOXM1的蛋白和mRNA水平以评价miR320对FOXM1的调控作用。克隆形成实验评价克隆形成能力；免疫荧光实验检测γH2AX荧光焦点数；Annexin ⅤFITC/PI双染法测定细胞凋亡；Western blotting测定XIAP、Bax、Bcl2和cleaved caspase3的表达水平；采用双荧光素酶报告基因实验验证miR320与FOXM1之间的靶向关系。
结果QPCR检测显示，Eca109细胞中miR320相对表达量最低，FOXM1相对表达量最高，故选取该细胞株进行后续实验。转染48 h后，转染组miR320表达量显著高于对照组；QPCR和Western blotting检测发现转染组FOXM1 mRNA和蛋白水平均低于对照组，差异均有统计学意义（P＜005）。与对照组相比，转染组Eca109细胞克隆形成能力显著降低，差异有统计学意义（P＜005）；转染组照射处理后1、6、24 h γH2AX焦点数增加，差异有统计学意义（P＜005）。转染组Eca109细胞的凋亡率高于对照组（P＜005）。与对照组比较，转染组Bax和cleaved caspase3表达增加，XIAP和Bcl2表达降低，差异有统计学意义（P＜005）。在野生型 FOXM13’UTR质粒转染细胞中，miR320 mimics转染组Eca109细胞的荧光素酶活性明显低于对照组（P＜005）；而在突变型质粒转染细胞中，两组细胞的荧光素酶活性的差异无统计学意义（P＞005）。
结论miR320能增强ESCC细胞放射敏感性，可能与靶向抑制FOXM1表达有关

关键词: 食管鳞癌, 放射敏感性, 微小RNA320（miR320）, 叉头框蛋白M1（FOXM1）

Abstract: ObjectiveTo investigate the mechanism of microRNA320（miR320） in regulating radiosensitivity in esophageal squamous cell carcinoma（ESCC） cells.
MethodsRealtime fluorescence quantitative PCR（QPCR） was used to detect the expressions of miR320 and forkhead box protein M1（FOXM1） in normal esophageal epithelial cells HEEC and ESCC cell lines of KYSE150， TE13 and Eca109. MiR320 mimics and its negative control （NC） were transfected into Eca109 cells by Lipofectamine 2000 and assigned to miR320 transfection group and control group， respectively. QPCR and Western blotting were used to detect the mRNA and protein levels of FOXM1 to evaluate the regulatory effect of miR320. Clone formation assay and immunofluorescence assay were performed to evaluate the cloneforming ability of Eca109 and the formation of γH2AX. The apoptotic rates of Eca109 cells were detected by flow cytometry and the expression levels of XIAP， Bax, Bcl2 and cleaved caspase3 were detected by Western blotting. Dual luciferase reporter assay was performed to verify the targeting relationship between miR320 and FOXM1.
ResultsCompared with HEEC cells， the expression of miR320 in Eca109 cells was the lowest， while the expression of FOXM1 was the highest（P＜005）， and Eca109 cells were chosen for the following experiments. Fortyeight hours after transfection， transfection group showed an increased miR320 expression compared with control group； and the mRNA and protein levels of FOXM1 in transfection group were both lower than control group（P＜005）. The colonyforming ability of Eca109 cells in transfection group was lower than that in control group（P＜005）.The number of γH2AX foci in transfection group at 1， 6 and 24 h after irradiation were significantly higher than those in control group（P＜005）. The apoptotic rate of Eca109 cells in transfection group was significantly higher than that in control group（P＜005）. Compared with control group， the expression of Bax and cleaved caspase3 increased and the expression of XIAP and Bcl2 were decreased in transfection group（P＜005）. The luciferase activity of Eca109 cells transfected with wildtype FOXM1 3’UTR plasmid and miR320 mimics was significantly lower than that of transfected with NC. No significant difference was observed between cells transfected with mutanttype FOXM1 3’UTR plasmid.
ConclusionmiR320 may enhance the radiosensitivity of ESCC cells and the underlying mechanism may associated with inhibition of FOXM1.

Key words: Esophageal squamous cell carcinoma, Radiosensitivity, MicroRNA320（miR320）, Forkhead box protein M1（FOXM1）

张潇文, 秦嗪, 孙新臣. 微小RNA320靶向FOXM1调控食管鳞癌放射敏感性的机制研究#br#[J]. 临床肿瘤学杂志, 2018, 23(6): 481-488.

ZHANG Xiaowen, QIN Qin, SUN Xinchen.. Effects of microRNA320 on the targeted regulation of FOXM1 and radiosensitivity of esophageal squamous cell carcinoma[J]. Chinese Clinical Oncology, 2018, 23(6): 481-488.

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