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Effect of shRNA mediated silencing of YB-1 protein on the expression of matrix collagenases in malignant melanoma cell in vitro

Ibrahim, Wisam Nabeel and Doolaanea, Abd Almonem and Abdull Rasad, Mohammad Syaiful Bahari (2018) Effect of shRNA mediated silencing of YB-1 protein on the expression of matrix collagenases in malignant melanoma cell in vitro. Cells, 7 (1). pp. 1-13. E-ISSN 2073-4409

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Abstract

Background and Objective: YB-1 is a transcription and oncogenic factor capable of binding to DNA and RNA performing versatile functions within normal and cancer cells. Some studies reported the binding of YB-1 with a collagenases gene promoter and influencing their expression. In addition, the role of YB-1 in malignant melanoma was not elucidated. Thus, in this study, the aim was to knock down the expression of YB-1 in A375 malignant melanoma cancer cell using the shRNA approach and study its effect on cancer cell proliferation, migration, and expression of collagenases. Methods: A375 malignant melanoma cell lines were grown in standard conditions and were transfected with three plasmids containing a retroviral pGFP-V-RS vector, two of them containing targeting sequences for YB-1 mRNA. The third plasmid contained a scrambled mRNA sequence as a negative control. Expression of YB-1 was validated using immune-fluorescence staining, RT-PCR and western blotting. The cancer cell proliferation was determined using MTT assay, serial trypan blue cell counting and cell cycle flow-cytometry analysis. Expression of collagenases (MMP1, MMP8, and MMP13) was evaluated using RT-PCR and western blotting analysis. In addition, a wound-healing assay was used to assess cell migration potential. Statistical analysis was performed using one-way ANOVA test with Bonferroni post hoc analysis to compare the quantitative results among samples. Results: The established silenced cell strains (P1 and P2) had nearly 70% knockdown in the expression of YB-1. These YB-1 silenced strains had a significant cell cycle-specific reduction in cell proliferation (p < 0.05 in serial cell counting and cell cycle flow cytometry analysis, p < 0.001 in MTT assay). In addition, YB-1 silenced strains had a remarkable reduction in cell migration potential. Expression of MMP13 was significantly reduced in YB-1 silenced strains. Conclusion: YB-1 oncoprotein is a promising target in the treatment of malignant melanoma. Silencing of this protein is associated with significant anti-proliferative, anti-invasive and MMP13 insulating properties in A375 malignant melanoma cancer cell lines.

Item Type: Article (Journal)
Additional Information: 6715/61453
Uncontrolled Keywords: YB-1; MMPs; collagenases; shRNA; malignant melanoma
Subjects: Q Science > Q Science (General)
Q Science > QP Physiology
R Medicine > RM Therapeutics. Pharmacology
Kulliyyahs/Centres/Divisions/Institutes (Can select more than one option. Press CONTROL button): Kulliyyah of Nursing
Depositing User: Dr Wisam Nabeel
Date Deposited: 22 Jan 2018 16:39
Last Modified: 24 Jan 2019 11:17
URI: http://irep.iium.edu.my/id/eprint/61453

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