Aberrant splicing of cdkn1a/p21 through micro-RNA mediated downregulation of JMJD6

Post-transcriptional modifications of tumor suppressors, including microRNA-mediated downregulation, are important events in tumor progression. To identify microRNAs involved in oncogenic transformation, we examined global microRNA profiles of three ras transgenic zebrafish models. Four microRNAs are upregulated in all transgenic systems. Analysis of the predicted targets shows that three of them target Jmjd6, and overexpression of Jmjd6 in ras transformed melanocytes blocks proliferation and melanoma development. We found that Jmjd6 functions to regulate splicing of the tumor suppressor cdkn1a/p21. Truncated cdkn1a/p21 transcripts, lacking the PCNA binding domain, accumulate in ras-expressing melanocytes during melanoma development. These findings implicate Jmjd6 in a novel mechanism for inactivation of a major tumor suppressor pathway in melanoma. A custom-designed Agilent zebrafish 8x15k miRNA platform was used to profile miRNA expression in transgenic zebrafish overexpressing the oncogene HRAS in melanocytes (kita:HRAS transgenic zebrafish (= Et(kita:Gal4TA, UAS:mCherry)hmz1;Tg(UAS:eGFP-HRASV12)io006, Santoriello et al., 2010) and in the telencephalon (zic:HRAS (= Et(zic4:Gal4TA4, UAS:mCherry)hzm5; Tg(UAS:eGFP-HRASV12)io006, Marina Mione, manuscript in preparation). The 15k design contained a duplicate of 7604 probes of 60-oligonucleotide length. The probes consisted of 2x22 nucleotide sequences antisense to mature miRNAs separated by a spacer of 8 nucleotides (CGATCTTT) and with a second spacer with the same sequence at the end. From 7604 probes 546 were designed for left (5') and right (3') arms of the hairpins of zebrafish miRNAs known in miRBase, while the remainder 7058 probes corresponded to predicted hairpin structures in the zebrafish genome that might include additional miRNAs but were not considered in this study. miRNA profiles of kita:HRAS transgenic zebrafish larvae were compared to control larvae at 3, 7, and 14 days post fertilization (dpf; 3 or 4 replicates per time point), miRNA profiles of zic:HRAS transgenic zebrafish larvae were compared to control larvae at 3 days post fertilization, and miRNA profiles of a heat-shock inducible RAS line (hsp70:HRAS = Tg(hsp70l:EGFP-HRASV12)i03, Santoriello et al., 2009) were analyzed at 6 hours after a 30 min 37 °C heat-shock induction at 3 dpf. In addition, miRNA profiles were determined of tumors in the tail fin of adult kita:HRAS fish and compared to tail fin tissue from control fish (3 replicates). For dual color hybridization of the Agilent chips miRNA samples from HRAS transgenics were labeled with Hy3 and samples from control fish were labeled with Hy5.