Supplementary Material for: MiR-199a-3p Overexpression Suppressed Cell Proliferation and Sensitized Chronic Myeloid Leukaemia Cells to Imatinib by Inhibiting mTOR Signalling

Introduction: Chronic myeloid leukaemia (CML) is a myeloproliferative neoplasm characterized by constitutive activity of the tyrosine kinase BCR-ABL1. Drug resistance remains one of the major challenges in CML therapy. MicroRNA (miR)-199a-3p plays an important role in many tumours but has rarely been investigated in CML. We aimed to analyse the role and mechanism of miR-199a-3p in regulating imatinib resistance in CML. Methods: The expression of miR-199a-3p and mammalian target of rapamycin (mTOR) in the serum of CML patients and CML cells was examined by qRT-PCR. The levels of apoptosis-related proteins were determined using western blot. The relative cell survival rate and cell proliferation were determined using a CCK-8 assay and bromodeoxyuridine (BrdU) assay, respectively. Cell cycle and apoptosis were analysed using flow cytometry. Moreover, a dual luciferase reporter assay was performed to verify the correlation between miR-199a-3p and mTOR. Results: MiR-199a-3p was downregulated in the serum of CML patients and in CML cells, while mTOR was upregulated. Both miR-199a-3p overexpression and mTOR silencing inhibited CML cell proliferation, promoted CML cell apoptosis and sensitized these cells to imatinib. mTOR silencing reversed the promoting effect of miR-199a-3p inhibition on the proliferation of CML cells and the inhibitory effects on cell apoptosis and sensitivity to imatinib. MiR-199a-3p directly targeted mTOR. Conclusion: MiR-199a-3p suppressed cell propagation, facilitated apoptosis of CML cells and sensitized CML cells to imatinib by downregulating mTOR signalling.

引言：慢性髓系白血病（Chronic myeloid leukaemia, CML）是一类以酪氨酸激酶BCR-ABL1组成性激活为特征的骨髓增殖性肿瘤。耐药仍是CML治疗的主要挑战之一。微小RNA（microRNA, miR）-199a-3p在多种肿瘤中发挥重要调控作用，但目前针对其在CML中的研究较为匮乏。本研究旨在探讨miR-199a-3p在调控CML细胞伊马替尼耐药中的作用及具体分子机制。
方法：采用实时定量聚合酶链反应（quantitative real-time polymerase chain reaction, qRT-PCR）检测CML患者血清及CML细胞中miR-199a-3p与哺乳动物雷帕霉素靶蛋白（mammalian target of rapamycin, mTOR）的表达水平；采用蛋白质印迹法（western blot）检测凋亡相关蛋白的表达水平；分别通过CCK-8实验与溴脱氧尿苷（bromodeoxyuridine, BrdU）实验检测细胞相对存活率与细胞增殖能力；采用流式细胞术分析细胞周期与细胞凋亡率；此外，通过双荧光素酶报告基因实验（dual luciferase reporter assay）验证miR-199a-3p与mTOR的靶向调控关系。
结果：miR-199a-3p在CML患者血清及CML细胞中均呈低表达，而mTOR则呈高表达。过表达miR-199a-3p或敲低mTOR均可抑制CML细胞增殖、促进细胞凋亡，并增强细胞对伊马替尼的敏感性。敲低mTOR可逆转抑制miR-199a-3p表达对CML细胞增殖的促进作用，以及对细胞凋亡率和伊马替尼敏感性的抑制效应。实验证实miR-199a-3p可直接靶向调控mTOR。
结论：miR-199a-3p通过下调mTOR信号通路，抑制CML细胞增殖、促进细胞凋亡，并增强CML细胞对伊马替尼的敏感性。