Krivosheeva Irina A, Filatova Alexandra Yu, Moshkovskii Sergei A, Baranova Ancha V, Skoblov Mikhail Yu
Laboratory of Functional Genomics, Research Centre of Medical Genetics, Erevanskaya Street, 10 building 2, Floor 44, Moscow, 115304 Russia.
Laboratory of Medical Proteomics, Institute of Biomedical Chemistry, Moscow, Russia.
Cancer Cell Int. 2020 Oct 7;20:488. doi: 10.1186/s12935-020-01584-2. eCollection 2020.
Cancers may be treated by selective targeting of the genes vital for their survival. A number of attempts have led to discovery of several genes essential for surviving of tumor cells of different types. In this work, we tried to analyze genes that were previously predicted to be essential for melanoma surviving. Here we present the results of transient siRNA-mediated knockdown of the four of such genes, namely, UNC45A, STK11IP, RHPN2 and ZNFX1, in melanoma cell line A375, then assayed the cells for their viability, proliferation and ability to migrate in vitro. In our study, the knockdown of the genes predicted as essential for melanoma survival does not lead to statistically significant changes in cell viability. On the other hand, for each of the studied genes, mobility assays showed that the knockdown of each of the target genes accelerates the speed of cells migrating. Possible explanation for such counterintuitive results may include insufficiency of the predicting computational models or the necessity of a multiplex knockdown of the genes.
To examine the hypothesis of essentiality of hypomutated genes for melanoma surviving we have performed knockdown of several genes in melanoma cell line and analyzed cell viability and their ability to migrate.
Knockdown was performed by siRNAs transfected by Metafectene PRO. The levels of mRNAs before and after knockdown were evaluated by RT-qPCR analysis. Cell viability and proliferation were assessed by MTT assay. Cell migration was assessed by wound healing assay.
The knockdown of the genes predicted as essential for melanoma survival does not lead to statistically significant changes in cell viability. On the other hand, for each of the studied genes, mobility assays showed that the knockdown of each of the target genes accelerates the speed of cells migrating.
Our results do not confirm initial hypothesis that the genes predicted essential for melanoma survival as a matter of fact support the survival of melanoma cells.
癌症可通过选择性靶向对其生存至关重要的基因来进行治疗。多项尝试已促使人们发现了多种不同类型肿瘤细胞生存所必需的基因。在这项研究中，我们试图分析先前预测对黑色素瘤生存至关重要的基因。在此，我们展示了在黑色素瘤细胞系A375中通过瞬时小干扰RNA（siRNA）介导敲低四个此类基因（即UNC45A、STK11IP、RHPN2和ZNFX1）的结果，然后检测了细胞的活力、增殖能力以及体外迁移能力。在我们的研究中，对预测为黑色素瘤生存所必需的基因进行敲低并未导致细胞活力出现统计学上的显著变化。另一方面，对于每个研究基因，迁移实验表明敲低每个靶基因都会加快细胞迁移速度。对此类与直觉相悖的结果的可能解释包括预测计算模型不够完善，或者需要对这些基因进行多重敲低。
为检验低突变基因对黑色素瘤生存至关重要这一假设，我们在黑色素瘤细胞系中敲低了多个基因，并分析了细胞活力及其迁移能力。
通过用Metafectene PRO转染的小干扰RNA进行敲低。通过逆转录定量聚合酶链反应（RT-qPCR）分析评估敲低前后的信使核糖核酸（mRNA）水平。通过MTT法评估细胞活力和增殖。通过伤口愈合实验评估细胞迁移。
对预测为黑色素瘤生存所必需的基因进行敲低并未导致细胞活力出现统计学上的显著变化。另一方面，对于每个研究基因，迁移实验表明敲低每个靶基因都会加快细胞迁移速度。
我们的结果并未证实最初的假设，即预测为黑色素瘤生存所必需的基因实际上支持黑色素瘤细胞的生存。
