Ibrahim Wisam Nabeel, Doolaanea Abd Almonem, Bin Abdull Rasad Mohammad Syaiful Bahari
Department of Basic Medical Sciences for Nursing, Faculty of Nursing, International Islamic University Malaysia, Kuantan Pahang 25200, Malaysia.
Department of Pharmaceutical Technology, Faculty of Pharmacy, International Islamic University Malaysia, Kuantan Pahang 25200, Malaysia.
Cells. 2018 Jan 10;7(1):7. doi: 10.3390/cells7010007.
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. 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. 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 ( < 0.05 in serial cell counting and cell cycle flow cytometry analysis, < 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. 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.
YB-1是一种转录和致癌因子，能够与DNA和RNA结合，在正常细胞和癌细胞中发挥多种功能。一些研究报道了YB-1与胶原酶基因启动子的结合及其对胶原酶表达的影响。此外，YB-1在恶性黑色素瘤中的作用尚未阐明。因此，在本研究中，目的是使用shRNA方法敲低A375恶性黑色素瘤癌细胞中YB-1的表达，并研究其对癌细胞增殖、迁移和胶原酶表达的影响。A375恶性黑色素瘤细胞系在标准条件下培养，并用三种含有逆转录病毒pGFP-V-RS载体的质粒进行转染，其中两种含有针对YB-1 mRNA的靶向序列。第三种质粒含有随机mRNA序列作为阴性对照。使用免疫荧光染色、RT-PCR和蛋白质印迹法验证YB-1的表达。使用MTT法、台盼蓝连续细胞计数和细胞周期流式细胞术分析测定癌细胞增殖。使用RT-PCR和蛋白质印迹分析评估胶原酶（MMP1、MMP8和MMP13）的表达。此外，使用伤口愈合试验评估细胞迁移潜力。使用单向方差分析和Bonferroni事后分析进行统计分析，以比较样本之间的定量结果。建立的沉默细胞株（P1和P2）的YB-1表达敲低了近70%。这些YB-1沉默株在细胞增殖方面具有明显的细胞周期特异性降低（连续细胞计数和细胞周期流式细胞术分析中P<0.05，MTT法中P<0.001）。此外，YB-1沉默株的细胞迁移潜力显著降低。在YB-1沉默株中，MMP13的表达显著降低。YB-1癌蛋白是恶性黑色素瘤治疗中有前景的靶点。该蛋白的沉默与A375恶性黑色素瘤细胞系中显著的抗增殖、抗侵袭和MMP13抑制特性相关。
