Xiong Ying, Liu Liqian, Qiu Ying, Liu Lili
Department of Dermatology, Linyi People's Hospital, Linyi, China.
Department of Pathology, Linyi People's Hospital, Linyi, China.
Cell Physiol Biochem. 2018;50(1):385-397. doi: 10.1159/000494015. Epub 2018 Oct 4.
BACKGROUND/AIMS: Melanoma is one of the most aggressive malignant tumors, with increasing incidence, poor prognosis, and lack of any effective targeted therapies. Abnormal expression of miR-29a has been found in several types of cancers, including melanoma. In this study, experiments were performed to investigate the role of miR-29a in melanoma, and the molecular mechanism by which miR-29a represses melanoma.
miR-29 and Bmi1 expression was examined by quantitative real-time polymerase chain reaction (qRT-PCR). The cell viability, apoptosis, migration and invasion were respectively determined by Cell Counting Kit-8 assay, Propidium iodide (PI) fluorescein isothiocynate (FITC)-Annexin V staining assay, wound healing assay and transwell assay. Luciferase reporter assay was performed to determine a target gene of miR-29a. Western blot was used to analyze protein expression of apoptosis-related proteins, Bmi1, Wnt/β-catenin and Nuclear factor-κB (NF-κB) pathway target genes.
miR-29a was down-regulated in all tested melanoma cell lines. Up-regulation of miR-29a effectively inhibited cell viability, migration, and invasion, but promoted apoptosis in A375 cells. Bmi1 was a direct target gene of miR-29a. Transfection with miR-29a mimic decreased cell migration and invasion and Bmi1 expression in Malme-3M cells, SK-MEL-2, SK-MEL-5, and M14 cell lines. Moreover, miR-29a might suppress growth, migration and invasion of A375 cells by negatively regulating Bmi1. In addition, our results demonstrated that transfection with miR-29a mimic effectively blocked Wnt/β-catenin and NF-κB pathways via down-regulating Bmi1.
miR-29a could be functioned as a potential tumor suppressor through direct regulation of Bmi1 in melanoma cells.
背景/目的：黑色素瘤是最具侵袭性的恶性肿瘤之一，其发病率不断上升，预后较差，且缺乏有效的靶向治疗方法。在包括黑色素瘤在内的多种癌症中均发现了miR-29a的异常表达。本研究通过实验探究miR-29a在黑色素瘤中的作用以及miR-29a抑制黑色素瘤的分子机制。
采用定量实时聚合酶链反应（qRT-PCR）检测miR-29和Bmi1的表达。分别通过细胞计数试剂盒-8法、碘化丙啶（PI）异硫氰酸荧光素（FITC）-膜联蛋白V染色法、伤口愈合试验和Transwell试验测定细胞活力、凋亡、迁移和侵袭能力。进行荧光素酶报告基因试验以确定miR-29a的靶基因。采用蛋白质印迹法分析凋亡相关蛋白、Bmi1、Wnt/β-连环蛋白和核因子-κB（NF-κB）信号通路靶基因的蛋白表达。
在所有检测的黑色素瘤细胞系中，miR-29a均呈下调表达。上调miR-29a可有效抑制A375细胞的活力、迁移和侵袭，但促进其凋亡。Bmi1是miR-29a的直接靶基因。转染miR-29a模拟物可降低Malme-3M细胞系、SK-MEL-2、SK-MEL-5和M14细胞中的细胞迁移和侵袭能力以及Bmi1表达。此外，miR-29a可能通过负向调节Bmi1抑制A375细胞的生长、迁移和侵袭。此外，我们的结果表明，转染miR-29a模拟物可通过下调Bmi1有效阻断Wnt/β-连环蛋白和NF-κB信号通路。
miR-29a可能通过直接调控黑色素瘤细胞中的Bmi1发挥潜在的抑癌作用。
