Department of Neuroscience, Biomedicine and Movement Sciences, Biology and Genetics Section, School of Medicine, University of Verona, I-37134 Verona, Italy.
Department of Neuroscience, Biomedicine and Movement Sciences, Biochemistry Section, School of Medicine, University of Verona, I-37134 Verona, Italy.
Int J Mol Sci. 2022 Jan 31;23(3):1647. doi: 10.3390/ijms23031647.
Onconase (ONC) is an amphibian secretory ribonuclease displaying cytostatic and cytotoxic activities against many mammalian tumors, including melanoma. ONC principally damages tRNA species, but also other non-coding RNAs, although its precise targets are not known. We investigated the ONC ability to modulate the expression of 16 onco-suppressor microRNAs (miRNAs) in the A375 BRAF-mutated melanoma cell line. RT-PCR and immunoblots were used to measure the expression levels of miRNAs and their regulated proteins, respectively. In silico study was carried out to verify the relations between miRNAs and their mRNA targets. A375 cell transfection with miR-20a-3p and miR-34a-5p mimics or inhibitors was performed. The onco-suppressors miR-20a-3p, miR-29a-3p and miR-34a-5p were highly expressed in 48-h ONC-treated A375 cells. The cytostatic effect of ONC in A375 cells was mechanistically explained by the sharp inhibition of cyclins D1 and A2 expression level, as well as by downregulation of retinoblastoma protein and cyclin-dependent-kinase-2 activities. Remarkably, the expression of kinases ERK1/2 and Akt, as well as of the hypoxia inducible factor-1α, was inhibited by ONC. All these proteins control pro-survival pathways. Finally, many crucial proteins involved in migration, invasion and metastatic potential were downregulated by ONC. Results obtained from transfection of miR-20a-3p and miR-34a-5p inhibitors in the presence of ONC show that these miRNAs may participate in the antitumor effects of ONC in the A375 cell line. In conclusion, we identified many intracellular downregulated proteins involved in melanoma cell proliferation, metabolism and progression. All mRNAs coding these proteins may be targets of miR-20a-3p, miR-29a-3p and/or miR-34a-5p, which are in turn upregulated by ONC. Data suggest that several known ONC anti-proliferative and anti-metastatic activities in A375 melanoma cells might depend on the upregulation of onco-suppressor miRNAs. Notably, miRNAs stability depends on the upstream regulation by long-non-coding-RNAs or circular-RNAs that can, in turn, be damaged by ONC ribonucleolytic activity.
ONC 是一种两栖分泌核糖核酸酶，对许多哺乳动物肿瘤，包括黑色素瘤，具有细胞抑制和细胞毒性作用。ONC 主要损伤 tRNA 物种，但也损伤其他非编码 RNA，尽管其确切靶标尚不清楚。我们研究了 ONC 调节 A375 BRAF 突变黑色素瘤细胞系中 16 个抑癌 microRNA（miRNA）表达的能力。RT-PCR 和免疫印迹分别用于测量 miRNA 和其调节蛋白的表达水平。进行了计算机研究以验证 miRNA 与其 mRNA 靶标之间的关系。进行了 A375 细胞的 miR-20a-3p 和 miR-34a-5p 模拟物或抑制剂的转染。在 48 小时 ONC 处理的 A375 细胞中高度表达抑癌因子 miR-20a-3p、miR-29a-3p 和 miR-34a-5p。ONC 在 A375 细胞中的细胞抑制作用可以通过 cyclin D1 和 A2 表达水平的急剧抑制以及视网膜母细胞瘤蛋白和细胞周期蛋白依赖性激酶-2 活性的下调来解释。值得注意的是，ONC 抑制了激酶 ERK1/2 和 Akt 以及缺氧诱导因子-1α的表达。所有这些蛋白均控制生存途径。最后，ONC 下调了许多参与迁移、侵袭和转移潜能的关键蛋白。在 ONC 存在下转染 miR-20a-3p 和 miR-34a-5p 抑制剂所获得的结果表明，这些 miRNA 可能参与了 ONC 在 A375 细胞系中的抗肿瘤作用。总之，我们鉴定了许多参与黑色素瘤细胞增殖、代谢和进展的细胞内下调蛋白。所有编码这些蛋白的 mRNAs 可能是 miR-20a-3p、miR-29a-3p 和/或 miR-34a-5p 的靶标，而这些 miRNA 又被 ONC 上调。数据表明，ONC 在 A375 黑色素瘤细胞中的几种已知的抗增殖和抗转移活性可能依赖于抑癌 miRNA 的上调。值得注意的是，miRNA 的稳定性取决于长非编码 RNA 或环状 RNA 的上游调节，而长非编码 RNA 或环状 RNA 又可以被 ONC 的核糖核酸酶活性破坏。
