MCPIP1 overexpression in human neuroblastoma cell lines causes cell‐cycle arrest by G1/S checkpoint block

Abstract

Monocyte chemoattractant protein-1-induced protein 1 (MCPIP1) has a multidomain structure, which assures its pleiotropic activity. The physiological functions of this protein include repression of inflammatory processes and the prevention of immune disorders. The influence of MCPIP1 on the cell cycle of cancer cells has not been sufficiently elucidated. A previous study by our group reported that overexpression of MCPIP1 affects the cell viability, inhibits the activation of the phosphoinositide-3 kinase/mammalian target of rapamycin signalling pathway, and reduces the stability of the MYCN oncogene in neuroblastoma (NB) cells.Furthermore, a decrease in expression and phosphorylation levels of cyclindependent kinase (CDK) 1, which has a key role in the M phase of the cell cycle, was observed. On the basis of these previous results, the purpose of our present study was to elucidate the influence of MCPIP1 on the cell cycle of NB cells. It was confirmed that ectopic overexpression of MCPIP1 in two human NB cell lines, KELLY and BE (2)-C, inhibited cell proliferation. Furthermore, flow cytometric analyses and imaging of the cell cycle with a fluorescence ubiquitination cell-cycle indicator test, demonstrated that overexpression of MCPIP1 causes an accumulation of NB cells in the G1 phase of the cell cycle, while the possibility of an increase in G0 phase due to induction of quiescence or senescence was excluded. Additional assessment of the molecular machinery responsible for the transition between the cell-cycle phases confirmed that MCPIP1 overexpression reduced the expression of cyclins A2, B1, D1, D3, E1, and E2 and decreased the phosphorylation of CDK2 and CDK4, as well as retinoblastoma protein. In conclusion, the present results indicated a relevant impact of overexpression of MCPIP1 on the cell cycle, namely a block of the G1/S cell-cycle checkpoint, resulting in arrest of NB cells in the G1 phase. K E Y W O R D S cell cycle, G1/S cell-cycle arrest, MCPIP1, neuroblastoma

MCPIP1 ribonuclease can bind and cleaveAURKAmRNA inMYCN-amplified neuroblastoma cells

“…In the present study, we elaborate on the mechanisms of MCPIP1 ribonuclease action in MNA neuroblastoma cells. Based on our previous findings [17][18][19], we proposed several putative MCPIP1's substrates in neuroblastoma cells. In order to verify their interaction with the ribonuclease, we demonstrated that the overexpression of MCPIP1 leads to decreased levels of a few mRNAs, encoding proteins facilitating cell cycle progression in a RNase-dependent manner.…”

“…The previously used experimental model contained the control for both, the MCPIP1 overexpression (cells transfected with an empty expression vector) and the protein's ribonucleolytic activity (cells overexpressing the mutated form of MCPIP1). Using this model we have shown a broad range of alteration in transcriptome and proteome of neuroblastoma cells overexpressing MCPIP1 protein via application of microarrays, RT-qPCR and western blot [17][18][19]31]. The decreased transcript expression in the cells overexpressing the wild-type form of MCPIP1, and simultaneously, unaltered levels in the cells with overexpression of the mutated form of the protein, points to the indispensability of the ribonucleolytic activity of MCPIP1 for the observed downregulation of a mRNA.…”

“…Elucidation of a ribonuclease action in cells requires the determination of its substrate RNAs. For better control of the ribonucleolytic activity of MCPIP1, we applied a point mutated form of the MCPIP1 protein (MCPIP1-D141N) instead of the deletion mutant, devoid of the ribonuclease domain, used by us so far [17][18][19]31]. Substitution of aspartic acid residue 141 with asparagine in the catalytic centre of the MCPIP1 protein results in the abolishment of ribonucleolytic activity, while the ability to bind specific RNAs is retained [9].…”

“…Cell division control protein 2 (CDC2), AURKA, Mammalian target of rapamycin (MTOR), Rapamycin-insenstivie companion of mTOR (RICTOR), Regulatory associated protein of mTOR (RPTOR), MYCN [17]; and CCND1, CCND3, CCNE1, CCNE2, Retinoblastoma (RB) [19]. The expression of the genes from this group was decreased in cells overexpressing MCPIP1-wt, but unaltered in cells overexpressing the deletion mutant of the protein in at least one of the studied neuroblastoma cell lines [17][18][19].…”

“…Cell division control protein 2 (CDC2), AURKA, Mammalian target of rapamycin (MTOR), Rapamycin-insenstivie companion of mTOR (RICTOR), Regulatory associated protein of mTOR (RPTOR), MYCN [17]; and CCND1, CCND3, CCNE1, CCNE2, Retinoblastoma (RB) [19]. The expression of the genes from this group was decreased in cells overexpressing MCPIP1-wt, but unaltered in cells overexpressing the deletion mutant of the protein in at least one of the studied neuroblastoma cell lines [17][18][19]. To estimate the probability of interaction between MCPIP1 and the candidate transcripts, we analysed the 3′UTRs to find evolutionarily conserved stemloop structures with pyrimidine-purine-pyrimidine sequences in the loop, as such structures were described as recognized by the MCPIP1 protein [9].…”