Non-coding RNA profiling in BRAFV600E-mutant cutaneous melanoma models before and after Spry1 depletion

Background: Drugs directly targeting the BRAF protein and the constitutive activation of the mitogen-activated protein kinase (MAPK) pathway have provided remarkable response rates in BRAFV600-mutant metastatic cutaneous melanoma (CM) patients. However, the occurrence of resistance persists, requiring a better understanding of molecular mechanisms underlying the treatment's response to improve therapeutic strategies. Grown evidence has linked microRNAs (miRNAs) to the biology and drug sensitivity of CM, as several of them have been demonstrated to modulate pathways driving tumor pathogenesis and immune response. Yet, the connection between specific miRNA signatures and CM-developed resistance to BRAF inhibitors (BRAFi) has not been completely elucidated. We have already demonstrated that Spry1, an upstream regulator of the MAPK signaling pathway, may represent a novel putative target for BRAFV600-mutant CM since Spry1 depletion, both in vitro and in vivo, reduces cell proliferation inducing cell cycle arrest and apoptosis that improves response to BRAFi vemurafenib. Methods: Here we performed a small RNA sequencing on three BRAFv600e-mutant metastatic CM cell lines stably engineered with Spry1 knock-down (Mel 593 Spry1KO clone 2, Mel 599 Spry1KO clone 9 and Mel 611 Spry1KO clone 4) to investigate the post-transcriptional gene regulation role of miRNAs in molecular and cellular functions modulation driven by loss of Spry1. Results: The classification of expressed sncRNA molecules revealed that, in all three clones, miRNAs were the most represented subclass, followed by small nuclear RNAs (snRNA), PIWI-interacting RNAs (piRNA), small nucleolar RNA (snoRNA) and RNA transfer (tRNA). We next compared miRNA profiles between Spry1KO clones and the parental cell lines to identify, among the clones, common and concordantly deregulated miRNAs which may have a role in the improved response to targeted therapy mediated by Spry1 depletion.

研究背景：直接靶向BRAF蛋白以及丝裂原活化蛋白激酶（mitogen-activated protein kinase, MAPK）通路组成型激活的药物，在BRAFV600突变型转移性皮肤黑色素瘤（cutaneous melanoma, CM）患者中已展现出显著的客观应答率。然而，耐药性的发生仍是临床亟待解决的难题，这要求我们更深入地阐明治疗应答背后的分子机制，以优化治疗策略。
越来越多的证据表明，microRNAs（miRNAs）与皮肤黑色素瘤的生物学特性及药物敏感性密切相关，已有多项研究证实这类非编码RNA可调控驱动肿瘤发生与免疫应答的信号通路。但目前，特定miRNA表达特征与皮肤黑色素瘤对BRAF抑制剂（BRAF inhibitors, BRAFi）耐药之间的关联尚未完全阐明。
我们此前的研究证实，作为丝裂原活化蛋白激酶信号通路的上游调控因子，Spry1或可成为BRAFV600突变型皮肤黑色素瘤的新型潜在治疗靶点：无论是在体外还是体内实验中，敲除Spry1均可抑制细胞增殖，诱导细胞周期阻滞与细胞凋亡，进而提升肿瘤细胞对BRAF抑制剂维莫非尼（vemurafenib）的应答效果。
研究方法：本研究针对3株经稳定敲除Spry1构建的BRAFV600E突变型转移性皮肤黑色素瘤细胞系（分别为Mel 593 Spry1KO克隆2、Mel 599 Spry1KO克隆9与Mel 611 Spry1KO克隆4）开展小RNA测序，旨在探究miRNAs在Spry1缺失所介导的分子与细胞功能调控中所发挥的转录后基因调控作用。
研究结果：对已表达的小非编码RNA（small non-coding RNA, sncRNA）分子进行分类后发现，3株克隆中miRNAs均为占比最高的亚型，其次分别为小核RNA（small nuclear RNA, snRNA）、PIWI互作RNA（PIWI-interacting RNA, piRNA）、小核仁RNA（small nucleolar RNA, snoRNA）与转运RNA（transfer RNA, tRNA）。随后，我们对比了Spry1敲除克隆与亲本细胞系的miRNA表达谱，以期在所有克隆中筛选出共同且表达趋势一致的差异miRNAs，这类miRNAs或参与Spry1缺失所介导的靶向治疗应答增强过程。