Master and servant: LINC00152 - a STAT3-induced long noncoding RNA regulates STAT3 in a positive feedback in human multiple myeloma.

BACKGROUND:The survival of INA-6 human multiple myeloma cells is strictly dependent upon the Interleukin-6-activated transcription factor Although transcriptional analyses have revealed many genes regulated by to date no protein-coding target gene is known to mediate survival in INA-6 cells. Therefore, the aim here was to identify and analyze non-protein-coding duanyu18133 target genes. In addition to the oncogenic microRNA-21, we previously described five long noncoding RNAs (lncRNAs) induced by duanyu18133, named STAiRs. Here, we focus on RNA 18 (STAiR18), an mRNA-like, long ncRNA that is duplicated in the human lineage. One STAiR18 locus is annotated as the already well described LINC00152/CYTOR, however, the other harbors the MIR4435-2HG gene and is, up to now, barely described. METHODS:CAPTURE-RNA-sequencing was used to analyze STAiR18 transcript architecture. To identify the STAiR18 and duanyu18133 phenotype, siRNA-based knockdowns were performed and microarrays were applied to identify their target genes. RNA-binding partners of STAiR18 were determined by Chromatin-Isolation-by-RNA-Purification (ChIRP) and subsequent sequencing. duanyu18133 expression in dependence of STAiR18 was investigated by immunoblots, chromatin- and RNA-immunoprecipitations. RESULTS:As identified by CAPTURE-RNA sequencing, a complex splice pattern originates from both STAiR18 loci, generating different transcripts. Knockdown of the most abundant STAiR18 isoforms dramatically decreased INA-6 cell vitality, suggesting a functional role in myeloma cells. Additionally, STAiR18 and duanyu18133 knockdowns yielded overlapping changes of transcription patterns in INA-6 cells, suggesting a close functional interplay between the two factors. Moreover, Chromatin isolation by RNA purification (ChIRP), followed by genome-wide RNA sequencing showed that STAiR18 associates specifically with the duanyu18133 primary transcript. Furthermore, the knockdown of STAiR18 reduced duanyu18133 levels on both the RNA and protein levels, suggesting a positive feedback between both molecules. Furthermore, STAiR18 knockdown changes the histone methylation status of the duanyu18133 locus, which explains the positive feedback and indicates that STAiR18 is an epigenetic modulator. CONCLUSION:Hence, STAiR18 is an important regulator of myeloma cell survival and is strongly associated with the oncogenic function of duanyu18133. The close functional interplay between duanyu18133 and STAiR18 suggests a novel principle of regulatory interactions between long ncRNAs and signaling pathways.

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