Long noncoding RNA ZFAS1 promoting small nucleolar RNA-mediated 2′-O-methylation via NOP58 recruitment in colorectal cancer
Long noncoding RNA ZFAS1 promoting small nucleolar RNA-mediated 2′-O-methylation via NOP58 recruitment in colorectal cancer
复制标题
DOI：
10.1186/s12943-020-01201-w
复制
发表时间：
2020-05-22
期刊：
影响因子：
37.3
通讯作者：
Wei, Minjie
中科院分区：
文献类型：
--
作者：
Wu, Huizhe;Qin, Wenyan;Wei, Minjie
越来越多的证据支持小核仁RNA（snoRNA）和长链非编码RNA（lncRNA）在表观遗传修饰水平上作为主基因调节子的作用。然而，这些功能性ncRNA在结直肠癌（CRC）中的潜在机制尚未得到很好的研究。方法采用基因芯片技术分析lncRNAs-snoRNAs-mRNAs的异常表达谱及其相关性和共表达富集情况。通过原位杂交（ISH）、RT-PCR、qPCR和免疫荧光（IF）检测候选lncRNA、snoRNA和靶基因。采用CCK 8、trans-well、细胞凋亡、IF法、western blot法和异种移植小鼠模型等体内、体外实验研究这些因子的生物学功能。通过RTL-P测定和基于单链立足点（DPBST）测定的新型双链引物来测定rRNA 2 '-O-甲基化（Me）活性。通过生物信息学、RNA稳定性、RNA荧光原位杂交、RNA下拉和翻译抑制等方法探讨了其分子机制。结果为了证明lncRNA和snoRNA参与了肿瘤发生过程中的2 '-O-Me修饰，我们发现了一个以前未报道的机制，该机制将ZFAS 1调控的snoRNP NOP 58控制SNORD 12 C，SNORD 78介导的rRNA 2 '-O-Me活性与CRC的发生和发展联系起来。具体而言，ZFAS 1发挥其致癌功能，并在CRC细胞和组织中显著上调，伴随着NOP 58、SNORD 12 C/78表达的升高。ZFAS 1基因敲减抑制了CRC细胞的增殖、迁移和细胞凋亡，并且这种抑制作用可以通过在体外和体内过表达NOP 58来逆转。从机制上讲，NOP 58蛋白可以被ZFAS 1的特异性基序（AAGA或CAGA）识别。该事件加速SNORD 12 C/78的组装以允许在相应的Gm 3878和Gm 4593位点进一步引导2 '-O-Me。重要的是，沉默SNORD 12 C或78降低了rRNA 2 '-O-Me活性，这可以通过过表达ZFAS 1来拯救，并且这随后抑制了其下游靶点（例如，EIF 4A 3和LAMC 2）。结论新的ZFAS 1-NOP 58-SNORD 12 C/78-EIF 4A 3/LAMC 2信号轴在结直肠癌发生中发挥作用，为进一步了解lncRNA-snoRNP介导的rRNA 2 '-O-Me活性在结直肠癌预防和治疗中的作用提供了新的思路。
Background 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. Methods 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. Results 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). Conclusion 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.