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浙江大学学报（农业与生命科学版）
浙江大学学报（农业与生命科学版）  2019, Vol. 45 Issue (5): 619-625    DOI: 10.3785/j.issn.1008-9209.2018.09.191
动物科学与动物医学     
人叉头转录因子O亚型6真核表达载体的构建及其对胶质瘤细胞存活的影响
龚小霞(),李超,曹曦月,杨诚诚,黄超()
四川农业大学动物医学院, 实验动物疾病模型研究室，成都 611130
Construction of eukaryotic expression vector of human forkhead box class O6 and its effect on glioma cell survival
Xiaoxia GONG(),Chao LI,Xiyue CAO,Chengcheng YANG,Chao HUANG()
Laboratory of Experimental Animal Disease Model, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 611130, China
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摘要：

构建人叉头转录因子O亚型6（forkhead box class O6, FOXO6）基因的真核表达载体，并探究FOXO6对胶质瘤细胞存活的影响。以人神经胶质瘤细胞U251的cDNA为模板，利用聚合酶链式反应（polymerase chain reaction, PCR）方法扩增FOXO6基因片段，并将其与pRK5-myc载体连接，构建pRK5-myc-FOXO6重组质粒；将构建成功的重组质粒和空载体分别转染至U251细胞中进行表达，通过实时荧光定量PCR（quantitative real-time PCR, qPCR）和4’,6-二脒基-2-苯基吲哚（4’, 6-diamidino-2-phenylindole, DAPI）染色法分别检测FOXO6对U251细胞中转录因子过氧化物酶体增殖物激活受体γ共激活因子1α（peroxisome proliferator-activated receptor γ coactivator-1α,PGC-1α）转录水平的影响及其对U251细胞存活的影响。经双酶切鉴定及DNA测序表明：pRK5-myc-FOXO6重组质粒构建成功；FOXO6转染组中的PGC-1α转录水平出现了明显的下降，并出现了明显的细胞凋亡现象。上述结果显示，pRK5-myc-FOXO6重组质粒构建成功，体外实验暗示FOXO6可能通过抑制PGC-1α的表达来抑制U251细胞的能量代谢及抗氧化过程，进而诱导U251细胞的凋亡，这为后续深入研究FOXO6在胶质瘤上的作用机制和靶点奠定了基础。
关键词： 人叉头转录因子O亚型6;  胶质瘤;  过氧化物酶体增殖物激活受体γ共激活因子1α    
Abstract:

To construct the eukaryotic expression vector of human forkhead box class O6 (FOXO6) and explore the effect of FOXO6 on glioma cell survival, we took the cDNA from glioma cell line U251 as a template, and the fragment of FOXO6 gene was amplified by polymerase chain reaction (PCR) and ligated with pRK5-myc vector to construct pRK5-myc-FOXO6 recombinant plasmid. Then, the pRK5-myc-FOXO6 recombinant plasmid and vacant vectors were transfected into U251 cells. The cell viability and the expression of transcription factor peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) were evaluated by 4’,6-diamidino-2-phenylindole (DAPI) staining and quantitative real-time PCR (qPCR), respectively. The result showed that the recombinant plasmid pRK5-myc-FOXO6 was successfully constructed by double enzyme digestion and DNA sequencing. The transcriptional level of PGC-1α in U251 cells showed a significant decrease after the FOXO6 transfection, and obvious apoptosis occurred after the FOXO6 overexpression. In conclusion, the eukaryotic expression vector of FOXO6 is constructed successfully. In vitro experiments suggest that FOXO6 may inhibit the energy metabolism and anti-oxidation process of U251 cells by inhibiting the expression of PGC-1α, and then induce the apoptosis of U251 cells. These results provide evidences for further in-depth studies exploring the mechanism and target of FOXO6 on glioma.
Key words: human forkhead box class O6    glioma    peroxisome proliferator-activated receptor γ coactivator-1α
收稿日期: 2018-09-19 出版日期: 2019-12-05
CLC:  Q 78  
基金资助: 国家自然科学基金(31501200)
通讯作者: 黄超     E-mail: gongxx0601@163.com;huangchao@sicau.edu.cn
作者简介: 龚小霞（https://orcid.org/0000-0002-6079-2121），E-mail：gongxx0601@163.com
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引用本文:

龚小霞,李超,曹曦月,杨诚诚,黄超. 人叉头转录因子O亚型6真核表达载体的构建及其对胶质瘤细胞存活的影响[J]. 浙江大学学报（农业与生命科学版）, 2019, 45(5): 619-625.

Xiaoxia GONG,Chao LI,Xiyue CAO,Chengcheng YANG,Chao HUANG. Construction of eukaryotic expression vector of human forkhead box class O6 and its effect on glioma cell survival. Journal of Zhejiang University (Agriculture and Life Sciences), 2019, 45(5): 619-625.

链接本文:

http://www.zjujournals.com/agr/CN/10.3785/j.issn.1008-9209.2018.09.191    或    http://www.zjujournals.com/agr/CN/Y2019/V45/I5/619

图1  PCR产物的琼脂糖凝胶电泳图M：DL5000分子标志物；A～C：PCR扩增产物。
图2  重组质粒pRK5-myc-FOXO6双酶切鉴定电泳图M：DL5000分子标志物；A～B：非阳性菌落；C～F：阳性菌落。
图3  重组质粒pRK5-myc-FOXO6的测序结果
图4  pRK5-myc-FOXO6蛋白表达的蛋白质印迹法检测1：转染pRK5-myc组；2：转染pRK5-myc-FOXO6组。n=3。
图5  U251细胞凋亡情况的DAPI染色检测箭头所指为凋亡的细胞核；n=4。
图6  空载体组和转染pRK5-myc-FOXO6重组质粒组的细胞凋亡率**表示在P＜0.01水平差异有高度统计学意义；n=4。
图7  PGC-1α表达情况的qPCR检测*表示在P＜0.05水平差异有统计学意义；n=4。
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