Long Non-Coding RNA MALAT1 Is Associated with the Progression of Multiple Myeloma and Induced By Cellular Stress
Authors: Kuroda Yuko , Kimura Kei , Masuda Yuta , Yamane Arito , Hattori Hikaru , Tahara Kenichi , Kaneko Ayaka , Suda Itsumi , Takahashi Noriyuki , Nanami Gotoh , Nagai Kie , Nagashima Tomomi , Takizawa Makiko , Koiso Hiromi , Ishizaki Takuma , Mitsui Takeki , Iriuchishima Hirono , Saitoh Takayuki , Shimizu Hiroaki , Yokohama Akihiko , Tsukamoto Norifumi , Kasamatsu Tetsuhiro , Minato Yusuke , Nojima Yoshihisa , Murakami Hirokazu , Handa Hiroshi 
Published: October 2019

Abstract
Background: 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 motility. Long non-coding RNA (lncRNAs), which are commonly defined as transcripts >200 nt in length, have emerged as a class of key regulatory RNA. LncRNAs are deregulated in diverse human cancers and associated with disease progression; however, little is known about its role in multiple myeloma (MM). To elucidate the role of lncRNAs in MM, we studied the expression patterns of several well-known lncRNAs in the plasma cells of MM, MGUS and plasmacytoma patients and the function in MM cell lines in vitro. Moreover, to reveal the distinct lncRNA signature comprehensively, we performed next-generation sequencing-based RNA sequencing.
Methods: CD138+ plasma cells from bone marrow (BM) mononuclear cells were obtained from 110 MM patients, 48 MGUS patients, 19 control subjects and 1 patient with extramedullary plasmacytoma of the liver and analyzed after obtaining informed consent from all the patients. The expression levels of lncRNAs MALAT1, ANRIL, HOTAIR, HOTTIP, and XIST were determined by a RQ-PCR analysis. RNase H-activating LNA™ GapmeR antisense oligonucleotides were used to knockdown lncRNA in vitro in MM cell lines. The cell lines were then treated with bortezomib, MG132, doxorubicin and hypoxic conditions to evaluate the effects of cytotoxic stress on the lncRNA expression. This study was approved by the IRB of Gunma University Hospital in accordance with the Declaration of Helsinki.
Results: A significant higher level of MALAT1 expression was observed in BM plasma cells of MM patients (4.49) compared to MGUS patients (1.51) and control subjects (0.55) (p<0.001). Strikingly, MALAT1 expression in extramedullary plasmacytoma of the liver was 140-fold higher compared with BM plasma cells obtained at the same time of sampling (433.7 vs 3.21). MALAT1 expression was higher in MM patients with t(4;14) and del 17p (10.05 vs 3.90, p=0.049; 5.22 vs 2.76, p=0.03, respectively), but no difference was observed between stages according to the International Staging System (ISS) (p=0.87). Neither the overall survival nor the progression-free survival differed between patients with high and low MALAT1 expression. ANRIL expression levels were diverse according to the patients (range, 0 to 294.3), however, the median expression was significantly higher in MM patients (p<0.001). HOTAIR and HOTTIP expression levels were not detected in most samples, and XIST expression was found only in female patient samples as expected. Interestingly, the MM cell lines KMS12PE, OPM2, KMS11 treated with bortezomib showed elevated MALAT1 expression by 4.3 -21.8 fold and ANRIL by 2.2-4.7 fold; however, this increase was not observed in bortezomib-resistant cell lines. Another proteasome inhibitor, MG132, and a low dose of the cytotoxic drug doxorubicin also elevated both lncRNAs in the cell lines. Hypoxic stress, which has been shown to induce MALAT1 in vascular cells, did not increase either lncRNA. MALAT1 knockdown by GapmeR did not affect cell proliferation. It has been shown that MALAT1 enhances cell motility of lung adenocarcinoma cells by influencing cell motility associated genes; however, the expression of previously reported affected genes, such as HMMR, CTHRC1 and ROD1, was not altered in the MALAT1 knockdown MM cell lines. Although t(4;14) was associated with a high MALAT1 expression in the patient samples, MMSET knockdown by siRNA did not change the MALAT1 expression in the cell lines, thus MMSET was not a regulator of MALAT1. RNA sequencing of MM and MGUS samples revealed a distinct lncRNA expression signature as well as protein coding genes.
Conclusion: Significant upregulation of lncRNAs MALAT1 and ANRIL might be associated with MM progression. Given that MALAT1 is associated with lung cancer metastasis, MALAT1 might be strongly associated with extramedullary plasmacytoma formation due to its high expression in liver plasmacytoma. Genotoxic and ER stress induced by therapeutic drugs might upregulate MALAT1 expression, leading to extramedullary extension, which is a recent problem in MM treatment. Determining the distinct lncRNA signature of MM is a current important issue to clarify the molecular mechanisms underlying MM progression for the development of novel therapies.

Disclosures
No relevant conflicts of interest to declare.