Differential microRNA Expression in USP8-Mutated and Wild-Type Corticotroph Pituitary Tumors Reflect the Difference in Protein Ubiquitination Processes

Background: USP8 mutations are the most common driver changes in corticotroph pituitary tumors. They have direct effect on cells' proteome through disturbance of ubiquitination process and also influence gene expression. The aim of this study was to compare microRNA profiles in USP8- mutated and wild-type tumors and determine the probable role of differential microRNA expression by integrative microRNA and mRNA analysis. Methods: Patients with Cushing's disease (n = 28) and silent corticotroph tumors (n = 20) were included. USP8 mutations were identified with Sanger sequencing. MicroRNA and gene expression was determined with next-generation sequencing. Results: USP8-mutated patients with Cushing's disease showed higher rate of clinical remission and trend towards lower tumor volume than wild-type patients. Comparison of microRNA profiles of USP8-mutated and wild-type tumors revealed 68 differentially expressed microRNAs. Their target genes were determined by in silico prediction and microRNA/mRNA correlation analysis. GeneSet Enrichment analysis of putative targets showed that the most significantly overrepresented genes are involved in protein ubiquitination-related processes. Only few microRNAs influence the expression of genes differentially expressed between USP8-mutated and wild-type tumors. Conclusions: Differences in microRNA expression in corticotropinomas stratified according to USP8 status reflect disturbed ubiquitination processes, but do not correspond to differences in gene expression between these tumors. Overall design: Pituitary tumor samples were collected during transsphenoidal surgery and fixed in formalin for routine diagnostic procedures, including immunohistochemical and ultrastruc- tural evaluation. Archival formalin-fixed paraffin-embedded (FFPE) tissue samples from 48 patients, including 28 samples from patients with Cushing's disease and 20 samples from patients with SCA, were included.

背景：泛素特异性蛋白酶8（USP8）突变是促肾上腺皮质激素细胞垂体瘤最常见的驱动变异。此类突变可通过扰动泛素化过程直接影响细胞蛋白质组，同时亦可调控基因表达。本研究旨在比较USP8突变型与野生型肿瘤的微小RNA（microRNA）表达谱，并通过整合微小RNA与信使RNA（mRNA）分析，明确差异表达微小RNA的潜在作用。
方法：本研究纳入库欣病患者28例、静默型促肾上腺皮质激素肿瘤患者20例。通过桑格（Sanger）测序鉴定USP8突变状态。采用下一代测序技术检测样本的微小RNA与基因表达水平。
结果：库欣病的USP8突变型患者临床缓解率更高，且肿瘤体积较野生型患者呈现降低趋势。对比USP8突变型与野生型肿瘤的微小RNA表达谱，共筛选得到68个差异表达的微小RNA。通过计算机预测与微小RNA-信使RNA关联分析，确定了这些差异表达微小RNA的靶基因。对推定靶基因开展基因集富集分析（Gene Set Enrichment Analysis），结果显示富集程度最高的基因集参与蛋白质泛素化相关生物学过程。仅有少量微小RNA可调控USP8突变型与野生型肿瘤间差异表达基因的转录水平。
结论：根据USP8状态分层的促肾上腺皮质激素瘤中，微小RNA表达差异反映了泛素化过程的紊乱，但与两类肿瘤间的基因表达差异并不具有对应性。
整体设计：垂体肿瘤样本经蝶窦手术采集后，以福尔马林固定以开展常规诊断流程，包括免疫组织化学检测与超微结构评估。本研究纳入48例患者的存档福尔马林固定石蜡包埋（FFPE）组织样本，其中28例来自库欣病患者，20例来自SCA患者。