miR-675 Mediates Downregulation of Twist1 and Rb in AFP-Secreting Hepatocellular Carcinoma
miR-675 Mediates Downregulation of Twist1 and Rb in AFP-Secreting Hepatocellular Carcinoma
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DOI：
10.1245/s10434-013-3106-3
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发表时间：
2013-12-01
影响因子：
3.7
通讯作者：
Shibata, D.
中科院分区：
文献类型：
--
作者：
Hernandez, J. M.;Elahi, A.;Shibata, D.
背景分泌甲胎蛋白（AFP）的肝细胞癌（HCC）代表了一种遗传学上不同的肿瘤亚型，通常与较差的预后相关。然而，这些表型差异背后的分子机制仍然知之甚少。来自27名患者的HCC肿瘤样品使用Affyester 133 Plus 2.0基因芯片进行分析。使用GeneGO Metacore软件鉴定改变的生物学途径。通过RT-PCR确认表达验证。通过过表达和抑制miR-675的表达，在软琼脂中检测miR-675对细胞周期、增殖、侵袭和生长的影响。我们确定了原发性肿瘤H19基因表达和血清AFP升高之间的密切关系。H19最近被鉴定为编码microRNA-675（miR-675），我们在一个独立的患者样本中证实了这种关系。对肝癌细胞中miR-675过表达的影响的途径分析显示，细胞粘附和细胞周期起始途径的显著上调。我们已经证明，miR-675介导的增殖和积累的细胞与四倍体DNA含量与Rb的抑制。我们还证明了miR-675的过表达改变了细胞形态，降低了侵袭潜力，并增加了锚定非依赖性生长能力。这些发现与间质向上皮的转变相一致，与关键EMT介质Twist 1表达的减少相关。miR-675在肝细胞癌中的表达将增殖和生长能力的显著上调与HCC细胞运动性的抑制联系起来。
Background. Alpha-fetoprotein (AFP)-secreting hepatocellular carcinomas (HCC) represent a genetically distinct subset of tumors often associated with a worse prognosis. However, the molecular mechanisms that underlie these phenotypic differences remain poorly understood.Methods. HCC tumor samples from 27 patients were profiled using the Affymetrix 133 Plus 2.0 GeneChips. GeneGO Metacore software was used to identify altered biologic pathways. Expression validation was confirmed by RT-PCR. Manipulation of miR-675 by overexpression and antagomir-mediated knockdown was carried out with subsequent evaluation of effects on cell behavior by cell cycle, proliferation, invasion, and growth in soft agar assays.Results. We identified a strong relationship between primary tumor H19 gene expression and elevated serum AFP. H19 has recently been identified to encode microRNA-675 (miR-675), and we confirmed the relationship in an independent sample of patients. Pathway analyses of the effect of miR-675 overexpression in hepatoma cells revealed a predominant upregulation of cell adhesion and cell cycle initiation pathways. We have demonstrated that miR-675 mediates increases in proliferation and an accumulation of cells with tetraploid DNA content associated with a repression of Rb. We also demonstrated that overexpression of miR-675 alters cellular morphology, reduces invasive potential, and increases anchorage-independent growth capacity. These findings are consistent with a mesenchymal-to-epithelial transition, associated with a reduction in the expression of the key EMT mediator, Twist1.Conclusions. Expression of the miR-675 in hepatocellular carcinoma links a dramatic upregulation of proliferative and growth capacity with inhibition of motility in HCC cells.