Płuciennik E, Nowakowska M, Gałdyszyńska M, Popęda M, Bednarek A K
Department of Molecular Carcinogenesis, Medical University of Lodz, 90-752 Lodz, Poland.
Department of Comparative Endocrinology, Medical University of Lodz, 90-752 Lodz, Poland.
Int J Mol Med. 2016 Mar;37(3):807-15. doi: 10.3892/ijmm.2016.2469. Epub 2016 Jan 27.
The purpose of the present study was to investigate the role of WW domain containing oxidoreductase (WWOX) downregulation in biological cancer-related processes in normal (non-malignant) and cancer endometrial cell lines. We created an in vitro model using the normal endometrial cell line, THESC, and 2 endometrial cancer cell lines with varying degrees of differentiation, the Ishikawa (well-differentiated) and the MFE296 (moderately differentiated) cells, in which the WWOX tumor suppressor gene was silenced using Gipz lentiviral shRNA. In this model, we examined the changes in invasiveness via biological assays, such as zymography, migration through a basement membrane, the adhesion of cells to extracellular matrix proteins, anchorage-independent growth and colony formation assay. We also evaluated the correlation between the mRNA expression of the WWOX gene and genes involved in the processes of carcinogenesis, namely catenin beta-1 (CTNNB1) and zinc finger E-box binding homeobox 1 (ZEB1) (gene transcription), cadherin 1 (CDH1) and ezrin (EZR) (cell adhesion), vimentin (VIM) (structural proteins), as well as phosphatase and tensin homolog (PTEN) (tumor suppression) and secreted protein, acidic, cysteine-rich (osteonectin) (SPARC) (SPARC) (cell growth regulation) by RT-qPCR. Downregulation of the WWOX gene in the moderately differentiated MFE296 cell line caused decreased migratory capacity, and a reduction of matrix metalloproteinase-2 (MMP-2) activity. However, these cells grew in semisolid medium and exhibited higher expression of CDH1 and EZR (cell adhesion) and secreted protein, acidic, cysteine-rich (osteonectin) (SPARC) (cell growth regulation). Moreover, in the well-differentiated endometrial cancer (Ishikawa) cell line, WWOX gene silencing resulted in an increased ability of the cells to proliferate indefinitely. Additionally, WWOX regulated changes in adhesion potential in both the normal and cancer cell lines. Our results suggest that the WWOX tumor suppressor gene modulated the processes of cell motility, cell adhesion, gene expression and remodeling in endometrial cell lines.
本研究的目的是探讨含WW结构域氧化还原酶（WWOX）下调在正常（非恶性）和癌性子宫内膜细胞系中与癌症相关生物学过程中的作用。我们利用正常子宫内膜细胞系THESC以及2种不同分化程度的子宫内膜癌细胞系——高分化的Ishikawa细胞系和中分化的MFE296细胞系建立了体外模型，在该模型中使用Gipz慢病毒shRNA使WWOX肿瘤抑制基因沉默。在这个模型中，我们通过生物学检测方法，如酶谱分析、穿过基底膜的迁移、细胞与细胞外基质蛋白的黏附、非锚定依赖性生长和集落形成试验，来检测侵袭性的变化。我们还通过RT-qPCR评估了WWOX基因的mRNA表达与参与致癌过程的基因之间的相关性，这些基因包括连环蛋白β-1（CTNNB1）和锌指E盒结合同源框1（ZEB1）（基因转录）、钙黏蛋白1（CDH1）和埃兹蛋白（EZR）（细胞黏附）、波形蛋白（VIM）（结构蛋白），以及磷酸酶和张力蛋白同源物（PTEN）（肿瘤抑制）和富含酸性半胱氨酸分泌蛋白（骨连接蛋白）（SPARC）（细胞生长调节）。在中分化的MFE296细胞系中，WWOX基因的下调导致迁移能力下降以及基质金属蛋白酶-2（MMP-2）活性降低。然而，这些细胞在半固体培养基中生长，并表现出较高的CDH1和EZR（细胞黏附）以及富含酸性半胱氨酸分泌蛋白（骨连接蛋白）（SPARC）（细胞生长调节）表达。此外，在高分化的子宫内膜癌（Ishikawa）细胞系中，WWOX基因沉默导致细胞无限增殖能力增强。此外，WWOX调节了正常和癌细胞系中黏附潜能的变化。我们的结果表明，WWOX肿瘤抑制基因调节了子宫内膜细胞系中的细胞运动、细胞黏附、基因表达和重塑过程。
