Tian R, Wang J, Yan H, Wu J, Xu Q, Zhan X, Gui Z, Ding M, He J
Department of Pathology, Anhui Provincial Hospital affiliated to Anhui Medical University and Anhui Provincial Cancer Hospital, Hefei, China.
Oncogene. 2017 Oct 19;36(42):5861-5873. doi: 10.1038/onc.2017.182. Epub 2017 Jun 19.
The function of miR16 in multiforme glioblastoma multiforme (GBM) and its stem cells (GSCs) remains elusive. To this end, we investigated the patterns of miR16 expression in these cells and their correlation with malignant behaviors and clinical outcomes. The levels of miR16 and its targeted genes in tumor tissue of GBM and GBM SGH44, U87, U251 cells as well as their stem cell counterparts were measured by qRT-PCR or western blot or immunohistochemistry. Luciferase reporter assay was used to confirm the binding of miR16 to 3'-UTR of its target genes. The effects of miR16 on malignant behaviors were investigated, including tumor cell viability, soft-agar colony formation, GSCs Matrigel colony forming and migration and invasion as well as nude mice xenograft model. Differentially expression patterns of miR16 in glioblastoma cells and GSCs cells were found in this study. Changes of miR16 targeted genes, Bcl2 (B cell lymphoma 2), CDK6 (Cyclin-dependent kinase 6), CCND1 (cyclin D1), CCNE1 (cyclin E1) and SOX5 were confirmed in glioblastoma cell lines and tissue specimens. In vitro and in vivo studies showed that tumor cell proliferation was inhibited by miR16 mimic, but enhanced by miR16 inhibitor. The expression level of miR16 positively correlates with GSCs differentiation, but negatively with the abilities of migration, motility, invasion and colony formation in glioblastoma cells. The inhibitory effects of miR16 on its target genes were also found in nude mice xenograft model. Our findings revealed that the miR16 functions as a tumor suppressor in GSCs and its association with prognosis in GBM.
miR16在多形性胶质母细胞瘤（GBM）及其干细胞（GSCs）中的功能仍不清楚。为此，我们研究了这些细胞中miR16的表达模式及其与恶性行为和临床结果的相关性。通过qRT-PCR、western blot或免疫组化检测GBM肿瘤组织以及GBM SGH44、U87、U251细胞及其干细胞对应物中miR16及其靶向基因的水平。采用荧光素酶报告基因检测法确认miR16与其靶基因3'-UTR的结合。研究了miR16对恶性行为的影响，包括肿瘤细胞活力、软琼脂集落形成、GSCs基质胶集落形成以及迁移和侵袭，还有裸鼠异种移植模型。本研究发现了胶质母细胞瘤细胞和GSCs细胞中miR16的差异表达模式。在胶质母细胞瘤细胞系和组织标本中证实了miR16靶向基因Bcl2（B细胞淋巴瘤2）、CDK6（细胞周期蛋白依赖性激酶6）、CCND1（细胞周期蛋白D1）、CCNE1（细胞周期蛋白E1）和SOX5的变化。体外和体内研究表明，miR16模拟物可抑制肿瘤细胞增殖，而miR16抑制剂则可增强肿瘤细胞增殖。miR16的表达水平与GSCs分化呈正相关，但与胶质母细胞瘤细胞的迁移、运动、侵袭和集落形成能力呈负相关。在裸鼠异种移植模型中也发现了miR16对其靶基因的抑制作用。我们的研究结果表明，miR16在GSCs中起肿瘤抑制作用，并且与GBM的预后相关。
