Downregulation of the PHLDA1 gene in IMR-32 neuroblastoma cells increases levels of Aurora A, TRKB and affects proteins involved in apoptosis and autophagy pathways.
We have recently shown that mRNA and protein of PHLDA1 (pleckstrin-homology-like domain family A, member  1) were by far the most upregulated molecules upon treatment of IMR-32 cells with the anti-GD2 ganglioside monoclonal antibody 14G2a. Hence, we decided to study functions of PHLDA1 using human neuroblastoma IMR-32 cells as a model. Here, we show that constitutive expression of mRNA and protein of the PHLDA1 gene in IMR-32 cells was inversely correlated with transcript of the AURKA gene and Aurora A oncoprotein. Next, we silenced PHLDA1 expression in IMR-32 cells using an shRNA interference method. We report that IMR-32 cells with stable downregulation of PHLDA1 showed enhanced cellular ATP levels and an increase in mitochondrial membrane potential, as compared to control and non-transduced cells. We demonstrated that downregulation of PHLDA1 leads to a significant increase in expression of Aurora A and TRKB that are markers of poor prognosis in neuroblastoma. Also, we measured an increase in Aurora A and Akt kinases phosphorylation in the cells. Most importantly, PHLDA1-silenced cells were less susceptible to apoptosis than control cells, as shown by the lower expression of cleaved caspase-3 and PARP as well as a decreased activity of caspase-3 and -7. Our study negatively correlates expression of PHLDA1 and Aurora A in IMR-32 cells and sheds new light on functions of PHLDA1 in the neuroblastoma tumor cells, suggesting its role as a pro-apoptotic protein. Additionally, our results show possible links of the protein to regulation of features of mitochondria and formation of autophagosomes.
Laboratory of Molecular Genetics and Virology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, 30-387 Kraków, Poland.
Małgorzata
M
Durbas
Laboratory of Molecular Genetics and Virology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, 30-387 Kraków, Poland.
Irena
I
Horwacik
Laboratory of Molecular Genetics and Virology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, 30-387 Kraków, Poland.
Elżbieta
E
Boratyn
Laboratory of Molecular Genetics and Virology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, 30-387 Kraków, Poland.
Hanna
H
Rokita
D000255:Adenosine Triphosphate / Q000096:biosynthesis; D017209:Apoptosis / Q000235:genetics; D064096:Aurora Kinase A / Q000096:biosynthesis* / Q000235:genetics; D001343:Autophagy / Q000235:genetics; D045744:Cell Line, Tumor; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D008562:Membrane Glycoproteins / Q000096:biosynthesis* / Q000235:genetics; D053078:Membrane Potential, Mitochondrial / Q000235:genetics; D008928:Mitochondria / Q000378:metabolism / Q000473:pathology; D009447:Neuroblastoma / Q000235:genetics* / Q000473:pathology; D011505:Protein-Tyrosine Kinases / Q000096:biosynthesis* / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis; D034741:RNA, Small Interfering / Q000235:genetics; D020813:Receptor, trkB; D014157:Transcription Factors / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics*
D008562:Membrane Glycoproteins; C491923:PHLDA1 protein, human; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D014157:Transcription Factors; D000255:Adenosine Triphosphate; D011505:Protein-Tyrosine Kinases; D020813:Receptor, trkB; C000591259:tropomyosin-related kinase-B, human; C576917:AURKA protein, human; D064096:Aurora Kinase A