Supplementary Material for: MicroRNA-Mediated Drug Repurposing Unveiled Potential Candidate Drugs for Prolactinoma Treatment

Introduction: Prolactinomas, also called lactotroph adenomas, are the most encountered type of hormone-secreting pituitary neuroendocrine tumors in the clinic. The preferred first-line therapy is a medical treatment with dopamine agonists (DAs), mainly cabergoline, to reduce serum prolactin levels, tumor volume, and mass effect. However, in some cases, patients have displayed DA resistance with aggressive tumor behavior or are faced with recurrence after drug withdrawal. Also, currently used therapeutics have notorious side effects and impair the life quality of the patients. Methods: Since the amalgamation of clinical and laboratory data besides tumor histopathogenesis and transcriptional regulatory features of the tumor emerges to exhibit essential roles in the behavior and progression of prolactinomas; in this work, we integrated mRNA- and microRNA (miRNA)-level transcriptome data that exploit disease-specific signatures in addition to biological and pharmacological data to elucidate a rational prioritization of pathways and drugs in prolactinoma. Results: We identified 8 drug candidates through drug repurposing based on mRNA-miRNA-level data integration and evaluated their potential through in vitro assays in the MMQ cell line. Seven repurposed drugs including 5-fluorocytosine, nortriptyline, neratinib, puromycin, taxifolin, vorinostat, and zileuton were proposed as potential drug candidates for the treatment of prolactinoma. We further hypothesized possible mechanisms of drug action on MMQ cell viability through analyzing the PI3K/Akt signaling pathway and cell cycle arrest via flow cytometry and Western blotting. Discussion: We presented the transcriptomic landscape of prolactinoma through miRNA and mRNA-level data integration and proposed repurposed drug candidates based on this integration. We validated our findings through testing cell viability, cell cycle phases, and PI3K/Akt protein expressions. Effects of the drugs on cell cycle phases and inhibition of the PI3K/Akt pathway by all drugs gave us promising output for further studies using these drugs in the treatment of prolactinoma. This is the first study that reports miRNA-mediated repurposed drugs for prolactinoma treatment via in vitro experiments.

引言：催乳素瘤（Prolactinomas），又称泌乳素腺瘤（lactotroph adenomas），是临床中最常见的分泌激素型垂体神经内分泌肿瘤。临床首选一线治疗方案为多巴胺激动剂（dopamine agonists, DAs）药物治疗，其中以卡麦角林（cabergoline）为主，用于降低血清催乳素水平、缩小肿瘤体积并减轻占位效应。然而，部分患者会出现多巴胺激动剂耐药，伴随肿瘤侵袭性行为，或在停药后出现肿瘤复发。此外，当前临床使用的治疗药物存在显著不良反应，会降低患者的生活质量。
方法：鉴于肿瘤的组织病理发生机制、转录调控特征与临床、实验室数据的整合，对催乳素瘤的生物学行为及疾病进展具有关键调控作用，本研究整合了mRNA与microRNA（miRNA）水平的转录组数据（该数据可反映疾病特异性分子特征），并结合生物学与药理学数据，以阐明催乳素瘤相关通路与药物的合理优先级排序。
结果：本研究通过mRNA-miRNA水平的多组学数据整合开展药物重定位研究，筛选得到8种候选药物，并通过MMQ细胞系的体外实验评估了其潜在活性。最终确定7种经重定位的药物可作为催乳素瘤治疗的潜在候选药物，分别为5-氟胞嘧啶（5-fluorocytosine）、去甲替林（nortriptyline）、来那替尼（neratinib）、嘌呤霉素（puromycin）、花旗松素（taxifolin）、伏立诺他（vorinostat）及齐留通（zileuton）。本研究进一步通过分析PI3K/Akt信号通路，并结合流式细胞术（flow cytometry）与蛋白质印迹法（Western blotting）检测的细胞周期阻滞情况，推测了候选药物对MMQ细胞存活能力的潜在作用机制。
讨论：本研究通过整合miRNA与mRNA水平的转录组数据，构建并呈现了催乳素瘤的转录组图谱，并基于该整合分析提出了经重定位的候选治疗药物。本研究通过检测细胞存活能力、细胞周期时相及PI3K/Akt蛋白表达水平，对上述研究结果进行了验证。所有候选药物对细胞周期时相的调控作用及对PI3K/Akt通路的抑制效果，均为后续将此类药物应用于催乳素瘤治疗的研究提供了极具前景的实验依据。本研究是首个通过体外实验报道基于miRNA调控的药物重定位方案用于催乳素瘤治疗的研究。