Chlorotoxin fusion protein regulates miR-374a and TNFAIP8 expression and inhibits glioma cell proliferation and promotes apoptosis

Yuan, Zhen; Zhang, Baihui; Zhuang, Jingling; Yang, Xu; Yu, Jiawen; Xu, Qiang; Azam, Nasir; ur Rahman, Khalil; Feng, Bin

doi:10.1007/s10616-020-00411-w

Original Article
Published: 19 July 2020

Chlorotoxin fusion protein regulates miR-374a and TNFAIP8 expression and inhibits glioma cell proliferation and promotes apoptosis

Zhen Yuan¹,
Baihui Zhang¹,
Jingling Zhuang¹,
Xu Yang¹,
Jiawen Yu²,
Qiang Xu³,
Nasir Azam¹,
Khalil ur Rahman¹ &
Bin Feng ORCID: orcid.org/0000-0002-3274-2143¹

Cytotechnology volume 72, pages 685–694 (2020)Cite this article

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Abstract

Glioblastoma multiforme is the most common primary central nervous system malignancy, accounting for half of all intracranial primary tumors. In this study we constructed a multifunctional chlorotoxin fusion protein E-CHP that combines enhanced green fluorescent protein (E), glioma-targeting peptide chlorotoxin (C), destabilizing lipid membrane peptide riHA2 (H), and C-terminal and mouse double minute domains of p53 (P). E-CHP was expressed in Escherichia coli and purified by His affinity chromatography. Fluorescence microscopy observation showed that E-CHP could effectively target glioma cells; real-time quantitative PCR revealed that E-CHP increased miR-374a expression; and the dual luciferase reporter assay showed that tumor necrosis factor alpha-induced protein (TNFAIP)8 is a direct target of miR-374a. E-CHP and miR-374a inhibited the proliferation and migration of glioma cells, and Western blot analysis indicated that they suppressed TNFAIP8 expression in glioma cells and promoted the expression of caspase-3 and -8. Finally, E-CHP and miR-374a stimulated the apoptosis of glioma cells, as determined by flow cytometry analysis. These results suggest that miR-374a is a new candidate target for glioma therapy, whereas E-CHP fusion protein has the potential to be developed as a multifunctional carrier for targeted drug delivery and therapy.

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Acknowledgements

This work was supported by the National Natural Science Foundation of China (Grant nos. 81371676, 81071248).

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Affiliations

Department of Biotechnology, College of Basic Medical Sciences, Dalian Medical University, Dalian, China
Zhen Yuan, Baihui Zhang, Jingling Zhuang, Xu Yang, Nasir Azam, Khalil ur Rahman & Bin Feng
Department of Hematology, The First Affiliated Hospital of Dalian Medical University, Dalian, China
Jiawen Yu
Faculty of Chemical, Environmental and Biological Science and Technology, Dalian University of Technology, Dalian, China
Qiang Xu

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Zhen Yuan
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Baihui Zhang
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Jingling Zhuang
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Xu Yang
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Jiawen Yu
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Qiang Xu
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Nasir Azam
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Khalil ur Rahman
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Bin Feng
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Correspondence to Bin Feng.

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Supplementary material 1 (DOCX 15 kb) Table S1 Primer sequences for plasmid construction and qRT-PCR.

Supplementary material 2 (JPEG 113 kb) Fig. S1 Schematic illustration of the E-CHP fusion protein.

Supplementary material 3 (JPEG 86 kb) Fig. S2 E-CHP upregulates miR-374a expression in U251 cells. *P < 0.05.

10616_2020_411_MOESM4_ESM.jpg

Supplementary material 4 (JPEG 222 kb) Fig. S3 Transwell experiments showing that the number of migrating U251 cells was reduced by miR-374a and E-CHP treatment compared to the control group. *P < 0.05.

10616_2020_411_MOESM5_ESM.jpg

Supplementary material 5 (JPEG 115 kb) Fig. S4 E-CHP and miR-374a inhibit TNFAIP8 mRNA expression in U251 cells. *P < 0.05, **P < 0.01.

Supplementary material 6 (JPEG 125 kb) Fig. S5 E-CHP and miR-374a inhibit TNFAIP8 protein expression in U251 cells. *P < 0.05.

10616_2020_411_MOESM7_ESM.jpg

Supplementary material 7 (JPEG 155 kb) Fig. S6 Caspase-3/8 expression in U251 cells was analyzed by Western blotting following E-CHP or miR-374a treatment. *P < 0.05.

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Yuan, Z., Zhang, B., Zhuang, J. et al. Chlorotoxin fusion protein regulates miR-374a and TNFAIP8 expression and inhibits glioma cell proliferation and promotes apoptosis. Cytotechnology 72, 685–694 (2020). https://doi.org/10.1007/s10616-020-00411-w

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Received: 09 January 2020
Accepted: 08 July 2020
Published: 19 July 2020
Issue Date: October 2020
DOI: https://doi.org/10.1007/s10616-020-00411-w

Keywords

Glioma
miR-374a
TNFAIP8
Chlorotoxin fusion protein
Targeted therapy