Department of Pharmacology, School of Pharmacy, China Medical University, Shenyang, 110122, People's Republic of China.
Liaoning Key Laboratory of molecular targeted anti-tumor drug development and evaluation; Liaoning Cancer immune peptide drug Engineering Technology Research Center; Key Laboratory of Precision Diagnosis and Treatment of Gastrointestinal Tumors, Ministry of Education, China Medical University, Shenyang, 110122, People's Republic of China.
Mol Cancer. 2020 May 22;19(1):95. doi: 10.1186/s12943-020-01201-w.
Increasing evidence supports the role of small nucleolar RNAs (snoRNAs) and long non-coding RNAs (lncRNAs) as master gene regulators at the epigenetic modification level. However, the underlying mechanism of these functional ncRNAs in colorectal cancer (CRC) has not been well investigated.
The dysregulated expression profiling of lncRNAs-snoRNAs-mRNAs and their correlations and co-expression enrichment were assessed by GeneChip microarray analysis. The candidate lncRNAs, snoRNAs, and target genes were detected by in situ hybridization (ISH), RT-PCR, qPCR and immunofluorescence (IF) assays. The biological functions of these factors were investigated using in vitro and in vivo studies that included CCK8, trans-well, cell apoptosis, IF assay, western blot method, and the xenograft mice models. rRNA 2'-O-methylation (Me) activities were determined by the RTL-P assay and a novel double-stranded primer based on the single-stranded toehold (DPBST) assay. The underlying molecular mechanisms were explored by bioinformatics and RNA stability, RNA fluorescence ISH, RNA pull-down and translation inhibition assays.
To demonstrate the involvement of lncRNA and snoRNAs in 2'-O-Me modification during tumorigenesis, we uncovered a previously unreported mechanism linking the snoRNPs NOP58 regulated by ZFAS1 in control of SNORD12C, SNORD78 mediated rRNA 2'-O-Me activities in CRC initiation and development. Specifically, ZFAS1 exerts its oncogenic functions and significantly up-regulated accompanied by elevated NOP58, SNORD12C/78 expression in CRC cells and tissues. ZFAS1 knockdown suppressed CRC cell proliferation, migration, and increased cell apoptosis, and this inhibitory effect could be reversed by NOP58 overexpression in vitro and in vivo. Mechanistically, the NOP58 protein could be recognized by the specific motif (AAGA or CAGA) of ZFAS1. This event accelerates the assembly of SNORD12C/78 to allow for further guiding of 2'-O-Me at the corresponding Gm3878 and Gm4593 sites. Importantly, silencing SNORD12C or 78 reduced the rRNAs 2'-O-Me activities, which could be rescued by overexpression ZFAS1, and this subsequently inhibits the RNA stability and translation activity of their downstream targets (e.g., EIF4A3 and LAMC2).
The novel ZFAS1-NOP58-SNORD12C/78-EIF4A3/LAMC2 signaling axis that functions in CRC tumorigenesis provides a better understanding regarding the role of lncRNA-snoRNP-mediated rRNAs 2'-O-Me activities for the prevention and treatment of CRC.
越来越多的证据支持小核仁 RNA(snoRNAs)和长非编码 RNA(lncRNAs)作为表观遗传修饰水平上的主要基因调控因子。然而，这些功能性 ncRNAs 在结直肠癌(CRC)中的潜在机制尚未得到很好的研究。
通过基因芯片微阵列分析评估 lncRNAs-snoRNAs-mRNAs 的失调表达谱及其相关性和共表达富集。通过原位杂交(ISH)、RT-PCR、qPCR 和免疫荧光(IF)检测候选 lncRNAs、snoRNAs 和靶基因。通过体外和体内研究包括 CCK8、trans-well、细胞凋亡、IF 检测、western blot 方法和异种移植小鼠模型来研究这些因素的生物学功能。通过 RTL-P 测定和基于单链结合物的双链引物(Double-stranded Primer Based on the single-stranded toehold, DPBST)测定来确定 rRNA 2'-O-甲基化(Me)活性。通过生物信息学和 RNA 稳定性、RNA 荧光原位杂交、RNA 下拉和翻译抑制测定来探索潜在的分子机制。
为了证明 lncRNA 和 snoRNA 参与肿瘤发生过程中的 2'-O-Me 修饰，我们揭示了一种以前未报道的机制，即 ZFAS1 调控的 snoRNP NOP58 控制 SNORD12C，SNORD78 介导的 rRNA 2'-O-Me 活性在 CRC 起始和发展中的作用。具体而言，ZFAS1 发挥其致癌功能，显著上调，并伴有 CRC 细胞和组织中 NOP58、SNORD12C/78 的表达升高。ZFAS1 敲低抑制 CRC 细胞增殖、迁移和增加细胞凋亡，而体外和体内过表达 NOP58 可逆转这种抑制作用。在机制上，NOP58 蛋白可被 ZFAS1 的特定基序(AAGA 或 CAGA)识别。这一事件加速了 SNORD12C/78 的组装，从而允许在相应的 Gm3878 和 Gm4593 位点进一步指导 2'-O-Me。重要的是，沉默 SNORD12C 或 78 降低了 rRNA 的 2'-O-Me 活性，而过表达 ZFAS1 可挽救这种活性，从而抑制其下游靶标(EIF4A3 和 LAMC2)的 RNA 稳定性和翻译活性。
在 CRC 肿瘤发生中发挥作用的新型 ZFAS1-NOP58-SNORD12C/78-EIF4A3/LAMC2 信号轴为了解 lncRNA-snoRNP 介导的 rRNA 2'-O-Me 活性在 CRC 的预防和治疗中的作用提供了更好的认识。
