Recent advances in high-throughput whole transcriptome analysis of human genome has allowed for the identifications of more than 13,000 long intergenic non-coding RNA transcripts (lincRNAs), with potential central roles in gene regulation and tumorigenesis. Using RNA-seq data from newly-diagnosed multiple myeloma (MM) patients, we identified linc00936 (chr 12) to be highly downregulated in CD138+ MM cells from 320 MM patients compared to 16 normal bone marrow plasma cells. Decreased expression of lincRNA was also observed in a panel of MM cells lines when compared with normal PBMC by both quantitative RT-PCR (qRT-PCR) and RNA-seq (N=66).
Interestingly, linc00936 overexpression significantly inhibited MM cell line growth (N=7), associated with G1 cell cycle arrest, decreased S phase, and induction of apoptotis via caspase 3 activation. Enforced expression of linc00936 was associated with induction of various gene sets, as measured by gene expression profiling. Importantly, linc00936 overexpression had a negative impact on genes involved in the mTOR pathway. WB analysis confirmed reduction in p-mTOR, along with a decrease in p-p70 S6 kinase and p-4E BP1, following linc00936 overexpression. Due to the role of mTOR pathway regulating autophagy, we next analyzed the effect of linc00936 on the autophagic response: autophagy was induced, evidenced by autophagic vacuoles and associated with decreased p62 and increased LC3A/B protein levels. Chloroquine, an autophagy inhibitor, partially abrogated the anti-MM activity of linc00936. RNA pull-down assay identified spleen tyrosine kinase (Syk), an E3 ubiquitin ligase (Rnf2), and serine/threonine-protein kinase (Taok3) as partner proteins. These interactions were validated by WB analysis of the lincRNA-pull-down proteins. Of note, the strongest interaction was with Syk, a well known modulator of mTOR activity. RNA-immunoprecipitation assay (RIP) followed by RT-PCR confirmed an increase in linc00936 levels in RNA-Syk complex compared to immunoglobulin G (IgG)-bound sample. Finally, treatment with demethylating agent 5-aza-deoxycitidine significantly increases the expression of linc00936 in RPMI-8226, MM1S and H929 MM cells, implicating promoter methylation as a mechanism of lincRNA suppression in MM. Based on these data, we analyzed the prognostic significance of linc00936 expression in MM patients enrolled on IFM/DFCI 2009 clinical study (N=296): low linc00936 expression was associated with significantly shorter event free survival compared to higher expression (p= 0.018). Collectively, our data provide the first evidence of a differentially expressed lincRNA in MM with a significant impact on tumor cell growth and survival via inhibition of mTOR pathway, with potential biological, prognostic and therapeutic implications.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.