ena-STUDY-Centrum Medyczne Ksztalcenia Podyplomowego-17-05-2017-12:56:22:355-6
Transcriptome analysis of clear cell renal carcinoma cell line overexpressing MCPIP1
Monocyte Chemoattractant Protein-induced protein 1 (MCPIP1) acts as a RNAse negatively regulating the expression of proinflammatory cytokines and transcription factors. We previously found that MCPIP1 overexpression downregulates the levels of transcripts encoding proteins induced in hypoxia (VEGF, GLUT1, IL-6). To investigate the full set of genes whose expression is regulated on MCPIP1-dependent manner, we performed RNA-Seq of clear cell renal cell carcinoma (ccRCC) Caki-1 cell line overexpressing wild-type (WT) MCPIP1 or its mutant form devoid of functional RNAse domain (D141N-MCPIP1). We identified 218 transcripts that were significantly downregulated in WT MCPIP1 when compared to D141N-MCPIP1 cells. We further validated the MCPIP1-dependent expression of 21 transcripts by quantitative RT-PCR analysis. Those transcripts encode regulators of the cell cycle, proteins induced in hypoxia and involved in signal transduction. Moreover, we found that WT MCPIP1 cells are characterised by elevated p21Cip1 protein level, which explains observed growth retardation phenotype. Elevated p21Cip1 levels in these cells may result from downregulation of one of the identified MCPIP1-dependent DDB1 transcript which encodes component of the Cul4-DDB1-Cdt2 E3 ligase important for proteolytic degradation of p21Cip1. Altogether, our results provide a further insight into mechanisms of MCPIP1 RNAse action in ccRCC cell line.
Monocyte Chemoattractant Protein-induced protein 1 (MCPIP1) acts as a RNAse negatively regulating the expression of proinflammatory cytokines and transcription factors. We previously found that MCPIP1 overexpression downregulates the levels of transcripts encoding proteins induced in hypoxia (VEGF, GLUT1, IL-6). To investigate the full set of genes whose expression is regulated on MCPIP1-dependent manner, we performed RNA-Seq of clear cell renal cell carcinoma (ccRCC) Caki-1 cell line overexpressing wild-type (WT) MCPIP1 or its mutant form devoid of functional RNAse domain (D141N-MCPIP1). We identified 218 transcripts that were significantly downregulated in WT MCPIP1 when compared to D141N-MCPIP1 cells. We further validated the MCPIP1-dependent expression of 21 transcripts by quantitative RT-PCR analysis. Those transcripts encode regulators of the cell cycle, proteins induced in hypoxia and involved in signal transduction. Moreover, we found that WT MCPIP1 cells are characterised by elevated p21Cip1 protein level, which explains observed growth retardation phenotype. Elevated p21Cip1 levels in these cells may result from downregulation of one of the identified MCPIP1-dependent DDB1 transcript which encodes component of the Cul4-DDB1-Cdt2 E3 ligase important for proteolytic degradation of p21Cip1. Altogether, our results provide a further insight into mechanisms of MCPIP1 RNAse action in ccRCC cell line.