Long Non-Coding RNA NEAT1 Is Upregulated By Heat Shock Factor 1 (HSF1) and Associated with Multiple Myeloma Progression
Authors: Watanabe Saki , Oda Tsukasa , Kuroda Yuko , Homma Kazuki , Murakami Yuki , Ishihara Rei , Yamane Eiko , Sunaga Masanobu , Kobayashi Nobuhiko , Osaki Yohei , Ishizaki Takuma , Shimizu Hiroaki , Koiso Hiromi , Iriuchishima Hirono , Takizawa Makiko , Yokohama Akihiko , Tsukamoto Norifumi , Nanami Gotoh , Ino Rumi , Saitoh Takayuki , Murakami Hirokazu , Handa Hiroshi 
Published: October 2019

Abstract
Background and Aims: Recent transcriptome-wide analyses have revealed an overwhelming amount of transcribed but not translated non-coding RNAs capable of influencing diverse cellular processes, such as proliferation, apoptosis, and cellular damage response. Long non-coding RNA (lnc RNA), which are commonly defined as transcripts >200 nt in length, have emerged as a class of key regulatory RNA. LncRNA are deregulated in diverse human cancers and associated with disease progression, however little is available in multiple myeloma (MM). We have previously shown that lnc RNA MALAT1 was a stress response gene associated with MM progression. We found that lnc RNA NEAT1 is also highly expressed in MM cells by transcriptome analysis with next generation sequencer (NGS). NEAT1 is recently revealed to play an important role on DNA damage response (DDR) as downstream of p53, and thereby involves in carcinogenesis. However its exact role in cancers is still in controversy. In this study, we tried to elucidate role and regulation mechanism of NEAT1 during MM progression.
Materials and Methods: Total 119 MM, 47 MGUS patients and 15 controls and 9 MM cell lines are subjected to the study after informed consent. The study was approved by IRB following Declaration of Helsinki. NEAT1 and its longer isoform NEAT1_2 RNA expressions were determined by RQ-PCR. RNA was extracted from purified CD138+ plasma cells from bone marrow (BM) mononuclear cells. The expression levels were normalized with ACTB and calculated with delta Ct value. Whole transcriptome analysis was performed in part of the samples by using Illumina Next Seq 500. MM cell lines transfected with tet-on p53 overexpression vector or tet-on sh-RNA HSF1 were used. RNase H-activating LNA™ GapmeR antisense oligonucleotides were used to knockdown lnc RNA in vitro.
Results: The expression level of NEAT1 was significantly higher in MM (median 0.97) than MGUS (median 0.31) (p<0.0001). NEAT1 level did not differ in between control (median 0.38) and MGUS (p=0.97). Although the median level was not statistically different (0.046 in MM; 0.031 in MGUS; 0.127 in control), substantial number of MM cases showed very high level of NEAT1_2. In MM samples, both NEAT1 and NEAT1_2 expression did not differ according to ISS (p=0.52, p=0.29) and cytogenetic risk group (p=0.49, p=0.203). NEAT1 and MALAT1 expression was positively correlated (r=0.632, p<0.0001 in all samples, r=0.62, p<0.0001 in MM only). NEAT1_2 was also positively correlated with MALAT1 expression (r=0.49, p<0.0001 in MM), and NEAT1 (r=0.35, p<0.0001 in MM). NEAT1 expression level and RNA structure were confirmed by transcriptome analysis with NGS. Since p53 promotes NEAT1/NEAT1_2 expression, we checked correlation in between these two genes expression levels. NEAT1 expression were positively correlated with both p53 and p21 (r=0.30, p<0.0001, r=0.41, p<0.0001). Positive correlations were also found in between NEAT1 and HSP90s (r=0.29, p=0.029 with HSP90AA1, r=0.29, p=0.029 with HSP90AB1, r=0.411, p=0.0018 with HSP90B1). NEAT1 was upregulated by MDM2 inhibitor nutlin3A in p53 wild type cell lines and by tet-on p53 overexpression in p53 null KMS11. Interestingly bortezomib and doxorubicin significantly increased NEAT1 and NEAT1_2 by 5-10 folds in MM cell lines even in p53 null KMS11. HSP90 inhibitors did not affect NEAT1/NEAT1_2 expression, but inhibition of HSF1, which is upstream transcription factor of HSP90, by either HSF1 inhibitor KNK437 or tet-on sh-HSF1 attenuated NEAT1/NEAT1_2 expression induced by bortezomib. NEAT1 knockdown by GapmeR did not affect cell growth. Overall survival and progression free survival of the newly diagnosed MM patients did not differ in between high and low NEAT1/NEAT1_2 expression.
Conclusion: Our results revealed that NEAT1/NEAT1_2 are regulated by heat shock pathway in addition to p53 pathway. Positive correlations of NEAT1 expression level with HSP90s level and existence of heat shock element in NEAT1 promoter region support this model. Considering the role of NEAT1/NEAT1_2 in DDR, our result suggests that this lncRNA may involve MM progression via damage response. Further studies elucidating roles of NEAT1 and other lncRNAs in MM contributes to development of novel therapy as well as to understand MM pathogenesis.

Disclosures
Tsukamoto: Kyowa-Kirin: Research Funding; Chugai: Research Funding; Eisai: Research Funding; Pfizer: Research Funding. Handa:Celgene: Honoraria, Research Funding, Speakers Bureau; Takeda: Consultancy, Honoraria, Research Funding, Speakers Bureau.