Identification of MiR-21-5p as a Functional Regulator of Mesothelin Expression Using MicroRNA Capture Affinity Coupled with Next Generation Sequencing
Identification of MiR-21-5p as a Functional Regulator of Mesothelin Expression Using MicroRNA Capture Affinity Coupled with Next Generation Sequencing
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DOI：
10.1371/journal.pone.0170999
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发表时间：
2017-01-26
期刊：
影响因子：
3.7
通讯作者：
Greene, Catherine M.
中科院分区：
文献类型：
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作者：
De Santi, Chiara;Vencken, Sebastian;Greene, Catherine M.
microRNA（miRNAs）是一类非编码小RNA，主要通过与3 '非翻译区（3' UTR）的miRNAs识别元件（MRE）结合沉默靶转录物来调控mRNA的表达。真正的目标的识别对于研究miRNA功能方面的研究人员来说是一个挑战。最近，我们开发了一种基于生物素化DNA反义寡核苷酸的方法（miR-CATCH），该方法捕获感兴趣的mRNA，并有助于在生理细胞环境中表征miRNA：：mRNA相互作用。在此，将miR-CATCH技术应用于间皮素（MSLN）基因，并与下一代测序（NGS）结合，以鉴定调节MSLN mRNA的miRNA，其可能是恶性胸膜间皮瘤（MPM）中发现的其蛋白水平增加的原因。使用生物素化的MSLN寡核苷酸从表达低水平MSLN的正常细胞系（Met-5A）中分离miRNA：：MSLN mRNA复合物。通过NGS鉴定靶向MSLN mRNA的miRNA，并选择miR-21- 5 p和miR-100- 5 p用于进一步验证分析。在一组恶性和非恶性细胞系的miRNA模拟实验中，miR-21- 5 p显示能够调节MSLN表达。在Mero-14细胞中的进一步的miRNA抑制剂实验和荧光素酶测定验证了miR-215 p作为MSLN的真正调节剂。此外，体外实验显示，用miR-21- 5 p模拟物处理降低了MPM细胞系的增殖。总之，这项工作表明，miR-CATCH技术，加上NGS和体外验证，代表了一种可靠的方法来确定天然miRNA：：mRNA的相互作用。MiR-21- 5 p被认为是MSLN的新调节剂，在细胞生长中具有可能的功能作用。
MicroRNAs (miRNAs) are small non-coding RNAs that regulate mRNA expression mainly by silencing target transcripts via binding to miRNA recognition elements (MREs) in the 3'untranslated region (3'UTR). The identification of bona fide targets is challenging for researchers working on the functional aspect of miRNAs. Recently, we developed a method (miR-CATCH) based on biotinylated DNA antisense oligonucleotides that capture the mRNA of interest and facilitates the characterisation of miRNAs:: mRNA interactions in a physiological cellular context. Here, the miR-CATCH technique was applied to the mesothelin (MSLN) gene and coupled with next generation sequencing (NGS), to identify miRNAs that regulate MSLN mRNA and that may be responsible for its increased protein levels found in malignant pleural mesothelioma (MPM). Biotinylated MSLN oligos were employed to isolate miRNA:: MSLN mRNA complexes from a normal cell line (Met-5A) which expresses low levels of MSLN. MiRNAs targeting the MSLN mRNA were identified by NGS and miR-21-5p and miR-100-5p were selected for further validation analyses. MiR-21-5p was shown to be able to modulate MSLN expression in miRNA mimic experiments in a panel of malignant and non-malignant cell lines. Further miRNA inhibitor experiments and luciferase assays in Mero-14 cells validated miR-215p as a true regulator of MSLN. Moreover, in vitro experiments showed that treatment with miR-21-5p mimic reduced proliferation of MPM cell lines. Altogether, this work shows that the miR-CATCH technique, coupled with NGS and in vitro validation, represents a reliable method to identify native miRNA:: mRNA interactions. MiR-21-5p is suggested as novel regulator of MSLN with a possible functional role in cellular growth.