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浙江大学学报（农业与生命科学版）
Journal of Zhejiang University (Agriculture and Life Sciences)  2019, Vol. 45 Issue (5): 619-625    DOI: 10.3785/j.issn.1008-9209.2018.09.191
Animal sciences & veterinary medicine     
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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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     
Received: 19 September 2018      Published: 05 December 2019
CLC:  Q 78  
Corresponding Authors: Chao HUANG     E-mail: gongxx0601@163.com;huangchao@sicau.edu.cn
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Xiaoxia GONG
Chao LI
Xiyue CAO
Chengcheng YANG
Chao HUANG
Cite this article:

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.

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


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

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 
Fig. 1 Agarose gel electrophoresis of PCR productsM: DL5000 DNA marker; A-C: Amplification products of PCR.
Fig. 2 Electrophoretogram of recombinant plasmid pRK5-myc-FOXO6 using double enzyme digestionM: DL5000 DNA marker; A-B: Non-positive colonies; C-F: Positive colonies.
Fig. 3 Sequencing results of recombinant plasmid pRK5-myc-FOXO6
Fig. 4 Detection of pRK5-myc-FOXO6 protein expression by Western blotting1: Transfection group of pRK5-myc; 2: Transfection group of pRK5-myc-FOXO6. n=3.
Fig. 5 Apoptotic detection of U251 cells by DAPI stainingThe apoptotic nuclei are shown at the arrows; n=4.
Fig. 6 Cell apoptosis rate in empty group and transfected pRK5-myc-FOXO6 recombinant plasmid groupDouble asterisks (**) indicate highly significant differences at the 0.01 probability level; n=4.
Fig. 7 Expression of PGC-1α detected by qPCRSingle asterisk (*) indicates significant differences at the 0.05 probability level; n=4.
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