Silencing expression of the NANOG gene and changes in migration and metastasis of urinary bladder cancer cells
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Submission date: 2014-09-23
Final revision date: 2014-12-18
Acceptance date: 2015-01-01
Online publication date: 2015-11-04
Publication date: 2016-06-30
Arch Med Sci 2016;12(4):889–897
Introduction: It 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-2I 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 methods: Human 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.
Results: The qRTPCR evaluation showed that silencing the NANOG gene in T24 cells led to the decrease of mRNA for the MMP-2I 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.
Conclusions: The 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-2I and MMP-9 expression levels.