Protein arginine methyltransferase 5 is a key regulator of the MYCN oncoprotein in neuroblastoma cells.
Bottom Line: Immunoblotting of NB cell-lines shows that high PRMT5 expression is strongly associated with MYCN-amplification (P < 0.004, Mann-Whitney U-test) and immunohistochemical analysis of primary NBs reveals that whilst PRMT5 protein is ubiquitously expressed in the cytoplasm of most cells, MYCN-amplified tumours exhibit pronounced nuclear PRMT5 staining.PRMT5 knockdown in MYCN-overexpressing cells, including the SHEP-21N cell-line with inducible MYCN expression leads to a dramatic decrease in MYCN protein and MYCN-associated cell-death in SHEP-21N cells.Together our studies implicate PRMT5 in a novel mode of MYCN post-translational regulation and suggest PRMT5 plays a major role in NB tumorigenesis.
Affiliation: Cancer Epigenetics Laboratory University of Bristol, Bristol BS8 1TD, UK.Show MeSH
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Mentions: We evaluated the effect of short-interfering RNA (siRNA) – mediated PRMT5 depletion on the SK-N-BE(2)C cell-line, representative of the poor prognosis subset of NB, together with two other HMTs known to be involved in cancer. Knockdown of CARM1 (PRMT4), EZH2 and PRMT5 all induced some cell death in SK-N-BE(2)C cells, but PRMT5 depletion triggered a significantly greater effect with approximately double the cell death compared to the negative control siRNA, and substantially greater death than either CARM1 or EZH2 knockdowns. Treatment of knockdowns with the caspase inhibitor Q-VD-OPh rescued cell death, and apoptosis was further confirmed by immunoblotting for poly(ADP-ribose) polymerase-1 (PARP-1) cleavage (Figure 1A–C). PRMT5 depletion also induced cell death in NGP cells, but not in SH-SY5Y NB cells or normal fibroblasts, despite efficient knockdowns (Figure 2 and Figure S1). PRMT5 depletion also had little effect on SHEP cells without high MYCN (see below). Increased cell death and PARP cleavage consistent with apoptosis was also apparent in NGP cells transfected with PRMT5 siRNA (Figure 2B and C).
Affiliation: Cancer Epigenetics Laboratory University of Bristol, Bristol BS8 1TD, UK.