Paul-Samojedny Monika, Pudełko Adam, Suchanek-Raif Renata, Kowalczyk Małgorzata, Fila-Daniłow Anna, Borkowska Paulina, Kowalski Jan
Department of Medical Genetics, School of Pharmacy with the Division of Laboratory Medicine, Medical University of Silesia, Jednosci 8 Street, 41-200, Katowice, Sosnowiec, Poland,
Tumour Biol. 2015 May;36(5):3263-77. doi: 10.1007/s13277-014-2955-0. Epub 2014 Dec 14.
Glioblastoma multiforme (GBM) is the most common primary brain malignancy, having a very poor prognosis and is characterized by extensive brain invasion as well as resistance to the therapy. The phosphoinositide 3-kinase (PI3K)/Akt/PTEN signaling pathway is deregulated in GBM. Besides, florid vascularization and aberrantly elevated vascular endothelial growth factor (VEGF) occur very often. The present study was designed to examine the inhibitory effect of AKT3, PI3KCA, and VEGFR2 small interfering RNAs (siRNAs) on GBM cell invasiveness. T98G cells were transfected with AKT3, PI3KCA, and/or VEGFR2 siRNAs. VEGFR2 protein-positive cells were identified by flow cytometry using specific monoclonal anti-VEGFR2 antibodies. Alterations in messenger RNA (mRNA) expression of VEGF, VEGFR2, matrix metalloproteinases (MMPs) (MMP-2, MMP-9, MMP-13, MMP-14), tissue inhibitors of metalloproteinases (TIMPs) (TIMP-1, TIMP-3), c-Fos, c-Jun, hypoxia-inducible factor-1α (HIF-1α), ObRa, and cathepsin D genes were analyzed by qRT-PCR. Cells treated with specific siRNA were also analyzed for invasion using the Matrigel invasion assay. We have found significantly lower mRNA levels of MMPs, cathepsin D, VEGF, VEGFR2, HIF-1α, and c-Fos/c-Jun ratio, as well as significantly higher mRNA level of TIMPs in AKT3 and PI3KCA siRNA transfected cells compared to untransfected cells, while significantly lower mRNA levels of MMPs (MMP-2, MMP-9, MMP-14) and TIMP-1, as well as significantly higher mRNA level of TIMP-3, were shown only in cells transfected with VEGFR2 siRNA. The positive correlation between MMP-13 and ObRa mRNA copy number has been found. Summarizing, transfection of T98G cells with AKT3, PI3KCA, or VEGFR2 siRNAs leads to a significant reduction in cell invasiveness. The siRNA-induced AKT3, PI3KCA, and VEGFR2 mRNA knockdown may offer a novel therapeutic strategy to reduce the invasiveness of GBM cells.
多形性胶质母细胞瘤（GBM）是最常见的原发性脑恶性肿瘤，预后极差，其特征是广泛的脑侵袭以及对治疗产生耐药性。磷脂酰肌醇3激酶（PI3K）/Akt/PTEN信号通路在GBM中失调。此外，GBM中常常出现丰富的血管生成以及血管内皮生长因子（VEGF）异常升高。本研究旨在检测AKT3、PI3KCA和VEGFR2小干扰RNA（siRNA）对GBM细胞侵袭性的抑制作用。用AKT3、PI3KCA和/或VEGFR2 siRNA转染T98G细胞。使用特异性抗VEGFR2单克隆抗体通过流式细胞术鉴定VEGFR2蛋白阳性细胞。通过qRT-PCR分析VEGF、VEGFR2、基质金属蛋白酶（MMPs）（MMP-2、MMP-9、MMP-13、MMP-14）、金属蛋白酶组织抑制剂（TIMPs）（TIMP-1、TIMP-3）、c-Fos、c-Jun、缺氧诱导因子-1α（HIF-1α）、ObRa和组织蛋白酶D基因信使核糖核酸（mRNA）表达的变化。还用基质胶侵袭试验分析用特异性siRNA处理的细胞的侵袭情况。我们发现，与未转染细胞相比，转染AKT3和PI3KCA siRNA的细胞中MMPs、组织蛋白酶D、VEGF、VEGFR2、HIF-1α的mRNA水平以及c-Fos/c-Jun比值显著降低，而TIMPs的mRNA水平显著升高；仅在转染VEGFR2 siRNA的细胞中，MMPs（MMP-2、MMP-9、MMP-14）和TIMP-1的mRNA水平显著降低，而TIMP-3的mRNA水平显著升高。已发现MMP-13与ObRa mRNA拷贝数之间呈正相关。总之，用AKT3、PI3KCA或VEGFR2 siRNA转染T98G细胞可导致细胞侵袭性显著降低。siRNA诱导的AKT3、PI3KCA和VEGFR2 mRNA敲低可能为降低GBM细胞的侵袭性提供一种新的治疗策略。
