Patrycja Przygodzka1, Izabela Papiewska-Pajak2, Helena Bogusz3, Jakub Kryczka4, Katarzyna Sobierajska5, M Anna Kowalska6, Joanna Boncela7. 1. Institute of Medical Biology, PAS, 106 Lodowa Street, 93232 Lodz, Poland. Electronic address: pprzygodzka@cbm.pan.pl. 2. Institute of Medical Biology, PAS, 106 Lodowa Street, 93232 Lodz, Poland. Electronic address: ipapiewska-pajak@cbm.pan.pl. 3. Institute of Medical Biology, PAS, 106 Lodowa Street, 93232 Lodz, Poland. Electronic address: hbogusz@cbm.pan.pl. 4. Institute of Medical Biology, PAS, 106 Lodowa Street, 93232 Lodz, Poland. Electronic address: jkryczka@cbm.pan.pl. 5. Department of Molecular Cell Mechanisms, Medical University, 6/8 Mazowiecka Street, 92215 Lodz, Poland. Electronic address: katarzyna.sobierajska@umed.lodz.pl. 6. Institute of Medical Biology, PAS, 106 Lodowa Street, 93232 Lodz, Poland; Department of Pediatrics, Children's Hospital of Philadelphia, Philadelphia, PA 19104, USA. Electronic address: kowalskam@email.chop.edu. 7. Institute of Medical Biology, PAS, 106 Lodowa Street, 93232 Lodz, Poland. Electronic address: jboncela@cbm.pan.pl.
Abstract
BACKGROUND: The epithelial-mesenchymal transition (EMT) is considered a core process that facilitates the escape of cancer cells from the primary tumor site. The transcription factor Snail was identified as a key regulator of EMT; however, the cascade of regulatory events leading to metastasis remains unknown and new predictive markers of the process are awaited. METHODS: Gene expressions were analysed using real-time PCR, protein level by Western immunoblotting and confocal imaging. The motility of the cells was examined using time-lapse microscopy. Affymetrix GeneChip Human Genome U133 Plus 2.0 analysis was performed to identify transcriptomic changes upon Snail. Snail silencing was performed using siRNA nucleofection. NMU detection was performed by ELISA. RESULTS: HT29 cells overexpressing Snail showed changed morphology, functions and transcriptomic profile indicating EMT induction. Changes in expression of 324 genes previously correlated with cell motility were observed. Neuromedin U was the second highest upregulated gene in HT29-Snail cells. This increase was validated by real-time PCR. Additionally elevated NMU protein was detected by ELISA in cell media. CONCLUSIONS: These results show that Snail in HT29 cells regulates early phenotype conversion towards an intermediate epithelial state. We provided the first evidence that neuromedin U is associated with Snail regulatory function of metastatic induction in colon cancer cells. GENERAL SIGNIFICANCE: We described the global, early transcriptomic changes induced through Snail in HT29 colon cancer cells and suggested NMU involvement in this process.
BACKGROUND: The epithelial-mesenchymal transition (EMT) is considered a core process that facilitates the escape of cancer cells from the primary tumor site. The transcription factor Snail was identified as a key regulator of EMT; however, the cascade of regulatory events leading to metastasis remains unknown and new predictive markers of the process are awaited. METHODS: Gene expressions were analysed using real-time PCR, protein level by Western immunoblotting and confocal imaging. The motility of the cells was examined using time-lapse microscopy. Affymetrix GeneChip Human Genome U133 Plus 2.0 analysis was performed to identify transcriptomic changes upon Snail. Snail silencing was performed using siRNA nucleofection. NMU detection was performed by ELISA. RESULTS: HT29 cells overexpressing Snail showed changed morphology, functions and transcriptomic profile indicating EMT induction. Changes in expression of 324 genes previously correlated with cell motility were observed. Neuromedin U was the second highest upregulated gene in HT29-Snail cells. This increase was validated by real-time PCR. Additionally elevated NMU protein was detected by ELISA in cell media. CONCLUSIONS: These results show that Snail in HT29 cells regulates early phenotype conversion towards an intermediate epithelial state. We provided the first evidence that neuromedin U is associated with Snail regulatory function of metastatic induction in colon cancer cells. GENERAL SIGNIFICANCE: We described the global, early transcriptomic changes induced through Snail in HT29 colon cancer cells and suggested NMU involvement in this process.
Authors: Katarzyna Sobierajska; Wojciech M Ciszewski; Marta E Wawro; Katarzyna Wieczorek-Szukała; Joanna Boncela; Izabela Papiewska-Pajak; Jolanta Niewiarowska; M Anna Kowalska Journal: Cells Date: 2019-08-01 Impact factor: 6.600