Intensive Care Unit, Hunan Cancer Hospital and The Affiliated Cancer Hospital of Xiangya Medical School, Central South University, Changsha, Hunan 410013, P.R. China.
Nuclear Medicine Department, Hunan Cancer Hospital and The Affiliated Cancer Hospital of Xiangya Medical School, Central South University, Changsha, Hunan 410013, P.R. China.
Mol Med Rep. 2021 May;23(5). doi: 10.3892/mmr.2021.11962. Epub 2021 Mar 24.
The development of novel treatments for lymphedema is hindered by the poorly understood pathophysiology of the disease. To improve the therapeutic success of treating the disease, the present study aimed to investigate the effects and mechanism of long non‑coding RNA myocardial infarction‑associated transcript (MIAT) in terms of the differentiation of adipose‑derived mesenchymal stem cells (ADMSCs) into lymphatic endothelial cells (LECs). The expression levels of (MIAT), microRNA (miR)‑495 and Prospero‑related homeobox 1 (Prox1) were measured by reverse transcription‑quantitative PCR. The protein expression levels of Prox1, lymphatic vessel endothelial hyaluronan receptor 1 (LYVE‑1), vascular endothelial growth factor receptor‑3 (VEGFR‑3) and podoplanin (PDPL) were detected by western blotting and immunofluorescence. A dual‑luciferase reporter assay was also used to detect the interaction between MIAT, miR‑495 and Prox1. In addition, migration and tube‑formation capabilities were measured by Transwell assay and tube‑formation assay, respectively. The results obtained demonstrated that VEGF‑C156S (recombinant VEGF‑C in which Cys156 was replaced by Ser residue) treatment could efficiently induce the differentiation of ADMSCs into LECs. MIAT expression was upregulated and miR‑495 was downregulated during differentiation. Mechanistically, MIAT upregulated Prox1 expression possibly by acting as a molecular sponge for miR‑495. Functional analyses indicated that the expression levels of Prox1, LYVE‑1, VEGFR‑3 and PDPL, and the migration and tube‑formation capabilities of ADMSCs induced by VEGF‑C156S, were significantly inhibited by silencing MIAT and overexpressing miR‑495. Moreover, miR‑495 inhibition and Prox1 overexpression reversed the effects of MIAT downregulation and miR‑495 upregulation, respectively, on the differentiation of ADMSCs into LECs. Taken together, these results suggested that MIAT may be involved in the differentiation of ADMSCs into LECs, and that the MIAT/miR‑495/Prox1 axis may be a novel regulatory mechanism and therapeutic target for the treatment of lymphedema.
新型淋巴管水肿治疗方法的发展受到该疾病病理生理学理解不足的阻碍。为了提高治疗疾病的治疗成功率，本研究旨在探讨长链非编码 RNA 心肌梗塞相关转录物（MIAT）在脂肪来源间充质干细胞（ADMSC）分化为淋巴管内皮细胞（LEC）方面的作用和机制。通过逆转录-定量 PCR 测量（MIAT）、微小 RNA（miR）-495 和 Prospero 相关同源框 1（Prox1）的表达水平。通过 Western blot 和免疫荧光检测 Prox1、淋巴管内皮透明质酸受体 1（LYVE-1）、血管内皮生长因子受体-3（VEGFR-3）和 podoplanin（PDPL）的蛋白表达水平。还使用双荧光素酶报告基因测定来检测 MIAT、miR-495 和 Prox1 之间的相互作用。此外，通过 Transwell 测定和管形成测定分别测量迁移和管形成能力。结果表明，VEGF-C156S（半胱氨酸 156 被丝氨酸取代的重组 VEGF-C）处理可有效诱导 ADMSC 分化为 LEC。在分化过程中，MIAT 表达上调，miR-495 表达下调。从机制上讲，MIAT 可能通过充当 miR-495 的分子海绵而上调 Prox1 的表达。功能分析表明，沉默 MIAT 和过表达 miR-495 显著抑制了 VEGF-C156S 诱导的 ADMSC 表达的 Prox1、LYVE-1、VEGFR-3 和 PDPL 的水平以及迁移和管形成能力。此外，miR-495 抑制和 Prox1 过表达分别逆转了 MIAT 下调和 miR-495 上调对 ADMSC 向 LEC 分化的影响。总之，这些结果表明，MIAT 可能参与 ADMSC 向 LEC 的分化，并且 MIAT/miR-495/Prox1 轴可能是治疗淋巴水肿的新的调节机制和治疗靶点。
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