Tilli Tatiana M, Bellahcène Akeila, Castronovo Vincent, Gimba Etel R P
Coordenação de Pesquisa, Programa de Carcinogênese Molecular, Instituto Nacional de Câncer (INCa)/Programa de Pós Graduação Stricto Sensu em Oncologia do INCa, Rio de Janeiro, RJ, Brazil.
BMC Cancer. 2014 Jun 13;14:433. doi: 10.1186/1471-2407-14-433.
Especially in human tumor cells, the osteopontin (OPN) primary transcript is subject to alternative splicing, generating three isoforms termed OPNa, OPNb and OPNc. We previously demonstrated that the OPNc splice variant activates several aspects of the progression of ovarian and prostate cancers. The goal of the present study was to develop cell line models to determine the impact of OPNc overexpression on main cancer signaling pathways and thus obtain insights into the mechanisms of OPNc pro-tumorigenic roles.
Human ovarian and prostate cancer cell lines, OvCar-3 and PC-3 cells, respectively, were stably transfected to overexpress OPNc. Transcriptomic profiling was performed on these cells and compared to controls, to identify OPNc overexpression-dependent changes in gene expression levels and pathways by qRT-PCR analyses.
Among 84 genes tested by using a multiplex real-time PCR Cancer Pathway Array approach, 34 and 16, respectively, were differentially expressed between OvCar-3 and PC-3 OPNc-overexpressing cells in relation to control clones. Differentially expressed genes are included in all main hallmarks of cancer, and several interacting proteins have been identified using an interactome network analysis. Based on marked up-regulation of Vegfa transcript in response to OPNc overexpression, we partially validated the array data by demonstrating that conditioned medium (CM) secreted from OvCar-3 and PC-3 OPNc-overexpressing cells significantly induced endothelial cell adhesion, proliferation and migration, compared to CM secreted from control cells.
Overall, the present study elucidated transcriptional changes of OvCar-3 and PC-3 cancer cell lines in response to OPNc overexpression, which provides an assessment for predicting the molecular mechanisms by which this splice variant promotes tumor progression features.
尤其是在人类肿瘤细胞中，骨桥蛋白（OPN）初级转录本会发生可变剪接，产生三种异构体，分别称为OPNa、OPNb和OPNc。我们之前证明，OPNc剪接变体可激活卵巢癌和前列腺癌进展的多个方面。本研究的目的是建立细胞系模型，以确定OPNc过表达对主要癌症信号通路的影响，从而深入了解OPNc促肿瘤发生作用的机制。
分别将人卵巢癌细胞系OvCar-3和前列腺癌细胞系PC-3稳定转染以过表达OPNc。对这些细胞进行转录组分析，并与对照进行比较，通过qRT-PCR分析确定基因表达水平和通路中依赖于OPNc过表达的变化。
使用多重实时PCR癌症通路阵列方法检测的84个基因中，与对照克隆相比，OvCar-3和PC-3 OPNc过表达细胞中分别有34个和16个基因差异表达。差异表达基因包括癌症的所有主要特征，并且使用相互作用组网络分析鉴定了几种相互作用蛋白。基于Vegfa转录本对OPNc过表达的显著上调，我们通过证明与对照细胞分泌的条件培养基（CM）相比，OvCar-3和PC-3 OPNc过表达细胞分泌的CM显著诱导内皮细胞粘附、增殖和迁移，部分验证了阵列数据。
总体而言，本研究阐明了OvCar-3和PC-3癌细胞系对OPNc过表达的转录变化，这为预测这种剪接变体促进肿瘤进展特征的分子机制提供了评估。
