Effect of shRNA mediated silencing of YB-1 protein on the expression of matrix collagenases in malignant melanoma cell in vitro
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 ro...
| Main Authors: | , , |
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| Format: | Article |
| Language: | English English |
| Published: |
MDPI
2018
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| Subjects: | |
| Online Access: | http://irep.iium.edu.my/61453/ http://irep.iium.edu.my/61453/ http://irep.iium.edu.my/61453/ http://irep.iium.edu.my/61453/1/61453_Effect%20of%20shRNA%20Mediated%20Silencing%20of%20YB-1%20Protein_ARTICLE.pdf http://irep.iium.edu.my/61453/2/61453%20Effect%20of%20shRNA%20Mediated%20Silencing%20of%20YB-1%20Protein%20on%20the%20Expression%20of%20Matrix%20Collagenases%20in%20Malignant%20Melanoma%20Cell%20In%20Vitro_WOS.pdf |
| Summary: | 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. |
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