Kandalam Moutushy Mitra, Beta Madhu, Maheswari Uma K, Swaminathan S, Krishnakumar Subramanian
Department of Ocular Pathology, Vision Research Foundation, Sankara Nethralaya, Chennai, Tamil Nadu, India.
Mol Vis. 2012;18:2279-87. Epub 2012 Aug 28.
Several miRNAs have been reported as candidate oncogenes and tumor suppressors, which are involved in the pathways specifically altered during tumorigenesis or metastasis. The miR 17-92 cluster located in 13q31 locus might contribute to retinoblastoma (RB) oncogenesis as 13q31 is amplified often in RB. We attempted to identify the factors involved in the regulation of miR 17-92 cluster in RB.
Real-time quantitative reverse transcriptase PCR was performed to study the expression of the miR 17-92 cluster in primary RB tumors and in Y79 cells after epithelial cell adhesion molecule (EpCAM) silencing. EpCAM was silenced using siRNA and confirmed by western blotting. The Y79 cells were transfected with individual and mixed antagomirs and studied the cell viability by (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, invasion by matrigel analysis and caspase-3 expression by flow cytometry.
The relative expression of miR 17-92 cluster, compared to that of a normal retina, ranged from 25 to 220 fold (p<0.0001), miR-18 being highly expressed in RB. Post EpCAM silencing resulted in a significant decrease (p<0.01) in the expression of the miR 17-92 cluster by 4 to eightfold in Y79 cells. Y79 cells transfected with an antagomirs mix (all 5 miRNAs) showed decreased cell viability (p<0.001) and cell invasion (p<0.001). Similarly, Y79 cells treated with antagomirs mix showed increased expression of caspase-3 (p<0.001), which confirms the anti-proliferative effect of antagomirs.
This study has showed varied expression of the miR17-92 cluster in primary RB tumors. EpCAM influences miR 17-92 cluster expression in retinoblastoma. In addition, we showed that the miR 17-92 cluster plays a role in RB cell proliferation and invasion. Therefore, targeting the miRNA 17-92 cluster may be beneficial for controlling Y79/RB cell proliferation and invasion.
已有报道称几种微小RNA（miRNA）为候选癌基因和肿瘤抑制因子，它们参与肿瘤发生或转移过程中特异性改变的信号通路。位于13q31位点的miR 17-92簇可能与视网膜母细胞瘤（RB）的肿瘤发生有关，因为13q31在RB中常发生扩增。我们试图确定参与RB中miR 17-92簇调控的因子。
采用实时定量逆转录聚合酶链反应研究原代RB肿瘤及上皮细胞黏附分子（EpCAM）沉默后的Y79细胞中miR 17-92簇的表达。使用小干扰RNA（siRNA）使EpCAM沉默，并通过蛋白质免疫印迹法进行确认。用单个和混合的抗miR转染Y79细胞，通过噻唑蓝（MTT）法研究细胞活力，通过基质胶分析研究细胞侵袭能力，并通过流式细胞术检测半胱天冬酶-3的表达。
与正常视网膜相比，miR 17-92簇的相对表达范围为25至220倍（p<0.0001），其中miR-18在RB中高表达。EpCAM沉默后，Y79细胞中miR 17-92簇的表达显著降低（p<0.01），降低了4至8倍。用抗miR混合物（所有5种miRNA）转染的Y79细胞显示细胞活力降低（p<0.001）和细胞侵袭能力降低（p<0.001）。同样，用抗miR混合物处理的Y79细胞显示半胱天冬酶-3的表达增加（p<0.001），这证实了抗miR的抗增殖作用。
本研究显示miR17-92簇在原代RB肿瘤中的表达存在差异。EpCAM影响视网膜母细胞瘤中miR 17-92簇的表达。此外，我们表明miR 17-92簇在RB细胞增殖和侵袭中起作用。因此，靶向miRNA 17-92簇可能有助于控制Y79/RB细胞的增殖和侵袭。
