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

Abstract

Background: The survival of INA-6 human multiple myeloma cells is strictly dependent upon the Interleukin-6activated transcription factor STAT3. Although transcriptional analyses have revealed many genes regulated by STAT3, to date no protein-coding STAT3 target gene is known to mediate survival in INA-6 cells. Therefore, the aim here was to identify and analyze non-protein-coding STAT3 target genes. In addition to the oncogenic microRNA-21, we previously described five long noncoding RNAs (lncRNAs) induced by STAT3, named STAiRs. Here, we focus on STAT3-induced 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 STAT3 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. STAT3 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 STAT3 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 STAT3 primary transcript. Furthermore, the knockdown of STAiR18 reduced STAT3 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 STAT3 locus, which explains the positive feedback and indicates that STAiR18 is an epigenetic modulator.

The noncoding RNA LINC00152 conveys contradicting effects in different glioblastoma cells

“…In previous studies we observed in Multiple Myeloma a positive feedback between LINC00152 and the transcription factor STAT3 at the transcriptional level. STAT3 induced the expression of LINC00152 lncRNA and vice versa LINC00152 raised the expression of STAT3 mRNA by a physical interaction with the STAT3 pre-mRNA 14 . To test whether such a positive feedback circle also exists in GBM cells, we performed knockdown experiments targeting STAT3 and LINC00152 by siRNAs.…”

“…Initially we aimed to determine whether the knockdown of LINC00152 in glioblastoma (GBM) results in the same anti-tumor phenotype as it has been observed in myeloma cells 14 . Since LINC00152 was also found to be overexpressed in GBM (Fig.…”

“…In previous studies, we found the long noncoding RNA LINC00C152 (also known as CYTOR, and STAiR18) to be overexpressed in a wide range of different tumor entities, which makes it a promising target for clinical use. Furthermore, we carried out a detailed analysis of LINC00C152 in Multiple Myeloma (MM) cells, discovering an impressive survival phenotype 13 , 14 . The Knockdown of LINC00152 dramatically decreased cell vitality in myeloma cells 14 .…”

“…Furthermore, we carried out a detailed analysis of LINC00C152 in Multiple Myeloma (MM) cells, discovering an impressive survival phenotype 13 , 14 . The Knockdown of LINC00152 dramatically decreased cell vitality in myeloma cells 14 . We showed that LINC00152 specifically associates with the STAT3 pre-mRNA and that the knockdown of LINC00152 reduced the transcription factor STAT3 at both, the RNA and protein level, suggesting a positive feedback between both molecules.…”

Circulating miR‑451a levels as a potential biomarker to predict the prognosis of patients with multiple myeloma

“…In terms of miR-451a, LINC00657 and AC084082.3 are shown to interact with miR-451a in BM ( 36 ). In MM, IL-6, a vital growth factor for myeloma cells, can induce the upregulation of several lncRNAs ( 37 ), and these lncRNAs may cause miR-451a dysregulation. However, which lncRNA can sponge miR-451a in MM remains unknown.…”