Silencing expression of the NANOG gene and changes in migration and metastasis of urinary bladder cancer cells

Abstract

IntroductionIt has been proved that expression of the NANOG gene is observed not only in embryonic-derived malignancies, but also in breast cancer, ovarian cancer, cervix cancer and bladder cancer. NANOG overexpression is correlated with high activity of MMP-2 and MMP-9. The aim of the study was to evaluate the changes in the malignant phenotype of T24 bladder cancer cells with modulated expression of the NANOG gene.Material and methodsHuman urinary bladder cancer cells T24 (HTB-4) were cultivated under standard conditions. Transfection of the cells with silencing constructions was performed with the application of Lipofectamine 2000 (Invitrogen) reagent. Evaluation of changes in the expression level of individual genes was performed using qRTPCR. Changes in the protein level were evaluated using the Human ELISA Kit (Abcam). The invasion capability of transfected cells was tested using Matrigel Invasion Chambers (BD Biosciences). The changes in cell migration were assessed with a wound-healing assay.ResultsThe qRTPCR evaluation showed that silencing the NANOG gene in T24 cells led to the decrease of mRNA for the MMP-2 gene to the level of 62.4% and the MMP-9 gene to the level of 76%. The cells with modulated expression of the NANOG gene migrated slower in the Matrigel invasion assay and in the wound-healing assay. The immunoenzymatic test showed a decrease in the protein level of MMP-9.ConclusionsThe transcriptional activity of the NANOG gene might be connected with some aspects of bladder cancer cell metastasis in vitro and has an influence on MMP-2 and MMP-9 expression levels.

Embracing study heterogeneity for finding genetic interactions in large‐scale research consortia

“…After filtering out the majority of irrelevant SNPs, we tested for interactions in the second stage. There were no SNPs reported in HaploReg with r > 0.8 2 to this SNP (in Europeans), and this SNP was thereby reported to affect the binding motif of NANOG gene, a transcription factor, that might be connected with some aspects of bladder cancer (Gawlik-Rzemieniewska, Galilejczyk, Krawczyk, & Bednarek, 2016;Mohd et al, 2017). The number of cases and controls in each genotype group for each pair of interacting SNPs is provided in Tables S1 and S2 of the supplementary materials.…”

“…We queried HaploReg v4.1 (Ward & Kellis, 2011) for evidence of enhancer activity or transcriptional factor blinding in cell lines at SNP rs1402938 (2 kb 3′ of the ANAPC1 gene) and at SNPs in high LD with this. There were no SNPs reported in HaploReg with r > 0.8 2 to this SNP (in Europeans), and this SNP was thereby reported to affect the binding motif of NANOG gene, a transcription factor, that might be connected with some aspects of bladder cancer (Gawlik-Rzemieniewska, Galilejczyk, Krawczyk, & Bednarek, 2016;Mohd et al, 2017). Furthermore, we queried GTEx (GTEx Consortium, 2015), a database of gene expression in normal human tissues, and found that ANAPC1 was expressed relatively ubiquitously, including in the normal human bladder (see Figure S1 in the supplementary materials).…”

Immune Curbing of Cancer Stem Cells by CTLs Directed to NANOG

“…NANOG also renderers CSCs resistant to chemotherapy, for example, by inhibition of p53-mediated apoptosis ( 47 ). Expression of NANOG and its pseudo genes is low or absent in normal cells, making it an ideal therapeutic target ( 48 – 51 ).…”

“…Expression of NANOG in CSCs is detected in a variety of cancer types, including glioma ( 34 ), breast cancer ( 52 ), ovarian cancer ( 53 ), and lung cancer ( 54 ). The realization that the resistant cancer cells driving tumor recurrence after classical treatment express NANOG ( 34 , 51 , 55 – 60 ) have prompted the development of NANOG inhibitors. These inhibitors were remarkably successful in experimental models ( 42 , 51 , 55 , 61 – 63 ).…”

CNTN-1 promotes docetaxel resistance and epithelial-to-mesenchymal transition via the PI3K/Akt signaling pathway in prostate cancer

“…As a dynamically reversible conversion cellular process, epithelial-mesenchymal transition (EMT) promotes the transformation of epithelial cells into mesenchymal cells in which mesenchymal cells acquire cell-cell adhesion, invasiveness and metastasis of the malignancy, thereby resulting in cancer cell mobility, anti-apoptosis abilities, and even poor prognosis [11][12][13][14]. EMT is central in embryogenesis, chronic inflammation, tissue remodeling, tumor metastasis, and multiple fibrotic diseases [15].…”