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Inhibition of cell proliferation, migration and invasion by a glioma-targeted fusion protein combining the p53 C terminus and MDM2-binding domain.

ABSTRACT: OBJECTIVES:The aim of this study was to develop multifunctional fusion proteins for targeting and delivering therapy elements into glioma cells. MATERIALS AND METHODS:Multifunctional fusion proteins were expressed in Escherichia coli and purified using Ni-NTA resin affinity chromatography. Human glioma cells and primary astrocytes were used to analyse their functions. Targeting proteins location to glioma cells was observed by confocal microscopy. Effects of cell viability and proliferation were evaluated using the Cell Counting Kit 8 and colony formation assays. Glioma cell migration and invasion were assessed using transwell assays, and apoptosis was analysed by flow cytometry. In addition, changes in expression of proteins related to the cell cycle and apoptosis were determined by Western blotting. RESULTS:The protein with highest bioactivity was GL1-riHA2-p53c+m-TAT (GHPc+mT), which combines glioma-targeting peptide GL1 (G), and C terminus (Pc) and mouse double minute domains (Pm) of p53, with the destabilizing lipid membrane peptide riHA2 (H) and cell-penetrating peptide TAT (T). The purified fusion protein was stable in cell culture medium and specifically targeted, and was internalized by, epidermal growth factor receptor (EGFR)-overexpressing glioma cells (U87?EGFR). It inhibited cell proliferation, migration and invasion, while flow cytometric analysis showed increased apoptosis. In addition, GHPc+mT caused significant changes in expression of proteins related to the cell cycle and apoptosis. CONCLUSION:GHPc+mT is a multifunctional protein combining targeting, inhibition of glioma cell proliferation and induction of apoptosis, providing some potential to be developed into an effective protein drug delivery system for glioma therapy.

SUBMITTER: Yu J

PROVIDER: S-EPMC6496415 | biostudies-literature | 2016 Feb

REPOSITORIES: biostudies-literature

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Inhibition of cell proliferation, migration and invasion by a glioma-targeted fusion protein combining the p53 C terminus and MDM2-binding domain.

Yu Jiawen J Guo Meihua M Wang Ting T Li Xiang X Wang Dan D Wang Xinying X Zhang Qian Q Wang Liang L Zhang Yang Y Zhao Chunhui C Feng Bin B

Cell proliferation 20160203 1

<h4>Objectives</h4>The aim of this study was to develop multifunctional fusion proteins for targeting and delivering therapy elements into glioma cells.<h4>Materials and methods</h4>Multifunctional fusion proteins were expressed in Escherichia coli and purified using Ni-NTA resin affinity chromatography. Human glioma cells and primary astrocytes were used to analyse their functions. Targeting proteins location to glioma cells was observed by confocal microscopy. Effects of cell viability and pro ...[more]

PMID: 26840447

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Project description:Long non‑coding RNAs (lncRNAs) have been implicated in the development and progression of tumors. However, the roles and underlying mechanisms of long intergenic non‑protein coding RNA 1116 (LINC01116), a member of the lncRNA family, in glioma progression are largely unclear. The expression of LINC01116 and microRNA (miR)‑744‑5p in glioma tissues and cells was detected by reverse transcription‑quantitative PCR. The influences of LINC01116 or miR‑744‑5p on cell proliferation and invasion were evaluated by Cell Counting Kit‑8, colony formation and Transwell assays, and western blotting was used to detect the expression of p53 pathway proteins. A dual‑luciferase reporter system was used to locate common binding sites between miR‑744‑5p and LINC01116 or the 3' untranslated region of E3 ubiquitin‑protein ligase Mdm2 (MDM2). RNA immunoprecipitation was used to determine the interactions between RNAs and proteins. Moreover, a xenograft mouse model was constructed to investigate the effects of LINC01116 in vivo, followed by a TdT‑mediated dUTP nick end labeling assay to determine the degree of apoptosis in nude mouse tumors. LINC01116 was found to be highly expressed in glioma tissues, which was associated with a malignant phenotype. LINC01116 promoted the proliferation and invasiveness of glioma cells, and inhibited the p53 pathway by preserving the expression of MDM2 mRNA via miR‑744‑5p sponging. Furthermore, a low degree of miR‑744‑5p expression was observed in glioma tissues, which was negatively associated with the expression of LINC01116. Overexpression of miR‑744‑5p inhibited the proliferation and invasiveness of glioma cells, which was rescued by LINC01116. Finally, LINC01116 knockdown inhibited tumor growth in nude mice. In conclusion, LINC01116 is aberrantly expressed and promotes the progression of glioma by regulating the miR‑744‑5p‑MDM2‑p53 pathway. In future, targeting LINC01116 may therefore be a potential therapeutic approach for patients with glioma.

Dataset Information

Inhibition of cell proliferation, migration and invasion by a glioma-targeted fusion protein combining the p53 C terminus and MDM2-binding domain.

Publications

Inhibition of cell proliferation, migration and invasion by a glioma-targeted fusion protein combining the p53 C terminus and MDM2-binding domain.

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