Weng Wen-Chin, Lin Kuan-Hung, Wu Pei-Yi, Lu Yi-Chien, Weng Yi-Cheng, Wang Bo-Jeng, Liao Yung-Feng, Hsu Wen-Ming, Lee Wang-Tso, Lee Hsinyu
Department of Pediatrics, College of Medicine, National Taiwan University Hospital and National Taiwan University, Taipei, Taiwan.
Mol Neurobiol. 2015 Aug;52(1):758-70. doi: 10.1007/s12035-014-8901-8. Epub 2014 Oct 7.
Calreticulin (CRT) has been previously correlated with the differentiation of neuroblastoma (NB), implying a favorable prognostic factor. Vascular endothelial growth factor (VEGF) has been reported to participate in the behavior of NB. This study investigated the association of CRT and VEGF-A in NB cells. The expressions of VEGF-A and HIF-1α, with overexpression or knockdown of CRT, were measured in three NB cells (SH-SY5Y, SK-N-DZ, and stNB-V1). An inducible CRT NB cell line and knockdown CRT stable cell lines were also established. The impacts of CRT overexpression on NB cell apoptosis, proliferation, and differentiation were also evaluated. We further examined the role of VEGF-A in the NB cell differentiation via VEGF receptor blockade. Constitutive overexpression of CRT led to NB cell differentiation without proliferation. Thus, an inducible CRT stNB-V1 cell line was generated by a tetracycline-regulated gene system. CRT overexpression increased VEGF-A and HIF-1α messenger RNA (mRNA) expressions in SH-SY5Y, SK-N-DZ, and stNB-V1 cells. CRT overexpression also enhanced VEGF-A protein expression and secretion level in conditioned media in different NB cell lines. Knockdown of CRT decreased VEGF-A and HIF-1α mRNA expressions and lowered VEGF-A protein expression and secretion level in conditioned media in different NB cell lines. We further demonstrated that NB cell apoptosis was not affected by CRT overexpression in stNB-V1 cells. Nevertheless, overexpression of CRT suppressed cell proliferation and enhanced cell differentiation in stNB-V1 cells, whereas blockage of VEGFR-1 markedly suppressed the expression of neuron-specific markers including GAP43, NSE2, and NFH, as well as TrkA, a molecular marker indicative of NB cell differentiation. Our findings suggest that VEGF-A is involved in CRT-related neuronal differentiation in NB. Our work may provide important information for developing a new therapeutic strategy to improve the outcome of NB patients.
钙网蛋白（CRT）先前已被证明与神经母细胞瘤（NB）的分化相关，这意味着它是一个有利的预后因素。据报道，血管内皮生长因子（VEGF）参与了NB的生物学行为。本研究调查了NB细胞中CRT与VEGF-A之间的关联。在三种NB细胞（SH-SY5Y、SK-N-DZ和stNB-V1）中检测了CRT过表达或敲低时VEGF-A和HIF-1α的表达。还建立了可诱导的CRT NB细胞系和敲低CRT的稳定细胞系。评估了CRT过表达对NB细胞凋亡、增殖和分化的影响。我们还通过VEGF受体阻断进一步研究了VEGF-A在NB细胞分化中的作用。CRT的组成型过表达导致NB细胞分化但不增殖。因此，通过四环素调控基因系统产生了可诱导的CRT stNB-V1细胞系。CRT过表达增加了SH-SY5Y、SK-N-DZ和stNB-V1细胞中VEGF-A和HIF-1α信使核糖核酸（mRNA）的表达。CRT过表达还增强了不同NB细胞系条件培养基中VEGF-A蛋白的表达和分泌水平。敲低CRT降低了不同NB细胞系条件培养基中VEGF-A和HIF-1α mRNA的表达，并降低了VEGF-A蛋白的表达和分泌水平。我们进一步证明，CRT过表达对stNB-V1细胞中的NB细胞凋亡没有影响。然而，CRT过表达抑制了stNB-V1细胞的增殖并增强了其分化，而阻断VEGFR-1则显著抑制了包括GAP43、NSE2和NFH在内的神经元特异性标志物以及TrkA（一种指示NB细胞分化的分子标志物）的表达。我们的研究结果表明，VEGF-A参与了NB中与CRT相关的神经元分化。我们的工作可能为开发改善NB患者预后的新治疗策略提供重要信息。
