Overexpression of miR-155-5p can upregulate antigen processing and presentation pathway via targeting tapasin

<strong>Background </strong>Dysregulation of major histocompatibility complex (MHC) class I antigen processing and presentation machinery (APM) components in the tumor as one main molecular mechanism of immune escape leading to deactivation of T cell immune surveillance could be due to post-transcriptional regulation via immune-modulatory microRNAs (miRNA). It is now well established from a variety of studies that several miRNAs could effectively modulate the expression of some MHC class I APM components in tumors. Tapasin is an important APM molecule involved in the association of MHC class I with transporter associated with antigen processing (TAP) and peptide loading. Since so far no detailed investigation of the posttranscriptional regulation of tapasin exists, the aim of this study is to identify and functionally characterize miRNAs targeting tapasin in melanoma. <strong>Methods </strong>Using miRNA trapping by RNA <em>in vitro</em> affinity purification (miTRAP) and <em>in silico </em>as well as small RNA sequencing, miRNAs will be identified, which bind to the 3'untranslated region (3' UTR) of tapasin. Dual luciferase assays will be performed to determine binding of the miRNA. <em>In silico</em> analysis was performed to predict the effect of miRNAs on the survival of melanoma patients in correlation to tapasin. RT-qPCR, Western blot, flow cytometry and other functional assays were performed after transfecting miRNA mimics in three melanoma cell lines. <strong>Results </strong>Using the combination strategy of miTRAP and RNA seq we identified miR-155-5p to bind to the 3’UTR of tapasin, which was further confirmed by <em>in silico analysis</em> and dual luciferase reporter assay. Transfection of miR-155-5p mimics demonstrated that miR-155-5p upregulate tapasin protein level, which was accompanied by an upregulation of the MHC class I (HLA-ABC) surface expression. Simultaneously, in several different types of cancer, including melanoma, the expression of miR-155-5p is significantly positively correlated with the patient's survival and HLA-A protein.研究背景 肿瘤中主要组织相容性复合体（major histocompatibility complex, MHC）I类抗原加工提呈机制（antigen processing and presentation machinery, APM）成分失调是导致T细胞免疫监视失活、进而引发免疫逃逸的核心分子机制之一，该失调可能由免疫调控性微小RNA（microRNAs, miRNA）介导的转录后调控所导致。现有多项研究已证实，多种miRNA可有效调控肿瘤中部分MHC I类APM成分的表达。伴珠蛋白（tapasin）是一类重要的APM分子，参与介导MHC I类分子与抗原加工相关转运体（transporter associated with antigen processing, TAP）的结合以及肽加载过程。截至目前，尚无针对tapasin转录后调控的系统性研究，因此本研究旨在鉴定黑色素瘤中靶向tapasin的miRNA，并对其功能进行表征。 研究方法 本研究通过基于RNA的体外（in vitro）亲和纯化miRNA捕获法（miRNA trapping by RNA in vitro affinity purification, miTRAP）、计算机辅助预测（in silico）分析及小RNA测序技术，筛选可结合tapasin 3'非翻译区（3' untranslated region, 3' UTR）的miRNA；采用双荧光素酶报告基因实验验证miRNA与靶序列的结合情况；通过in silico分析预测miRNA的表达与黑色素瘤患者生存率及tapasin表达的相关性；在三株黑色素瘤细胞系中转染miRNA模拟物（miRNA mimics）后，开展实时定量聚合酶链式反应（real-time quantitative polymerase chain reaction, RT-qPCR）、蛋白质免疫印迹（Western blot）、流式细胞术（flow cytometry）及其他功能学实验。 研究结果 本研究通过整合miTRAP与RNA测序的联合筛选策略，鉴定出miR-155-5p可结合tapasin的3' UTR，该结果经in silico分析及双荧光素酶报告基因实验进一步验证。转染miR-155-5p模拟物后，tapasin的蛋白表达水平显著上调，同时伴随MHC I类（HLA-ABC）分子的表面表达升高。此外，在包括黑色素瘤在内的多种癌症类型中，miR-155-5p的表达水平与患者生存率及HLA-A蛋白表达均呈显著正相关。