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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This work was supported by the National Natural Science Foundation of China (Grant nos. 81371676, 81071248).
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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.
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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.
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Supplementary material 6 (JPEG 125 kb) Fig. S5 E-CHP and miR-374a inhibit TNFAIP8 protein expression in U251 cells. *P < 0.05.
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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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DOI: https://doi.org/10.1007/s10616-020-00411-w