miRNAs are non-coding small RNAs that modulate protein expression at the post-transcriptional level and are implicated in the pathogenesis of a variety of cancers. In Multiple Myeloma (MM) a global elevation of miRNAs was previously correlated with poor disease outcomes and response to therapy. Using miRNome profiling of MM patients, we have recently established a miRNA-based risk score that is predictive of response to lenalidomide (Neri P, Blood 2011). In particular, we identified significant upregulation of miR-30 family members (a, b, c and e) in lenalidomide resistant patients. In the present study, we evaluated the biological functions of miR-30e in MM and its role in plasma cells resistance to lenalidomide as well as other anti-MM therapeutics.
Microarray profiling (Affymetrix miRNA GeneChip) of total RNA extracted from bone marrow plasma cells from lenalidomide sensitive and resistant MM patients (n=40), coupled with quantitative short stem-loop PCR (TaqMan, Applied Biosystems), confirmed the upregulation of miR-30e in lenalidomide resistant patients. Functionally, we sought to determine if overexpression of miR-30e would modify MM cells sensitivity to lenalidomide and bortezomib. Lentiviral-mediated stable expression (pLKO.1 retroviral plasmid) of miR-30e, and relative to empty vector (EV), significant increased MM1S and OPM2 cells growth (1.3 fold) as determined by MTT assay. In addition, miR-30e overexpressing cells (MM1S-30e and OPM2-30e vs MM1-EV and OPM2-EV) were more resistant to the cytotoxic effects of lenalidomide as well as bortezomib with approximately 15 to 20% reduction in cells death (Annexin V staining and MTT assay). Computational target prediction analysis (TargetScan 6.0 and miRanda) identified CRBN and BLIMP1 as potential target of miR-30e with a miRNA seed region that matches 8 or 7mer sites within Cereblon and BLIMP1 3'UTR regions. In a panel of MM cell lines (MM1S, OPM2, H929, INA-6, U266, 8226, KMS11) CRBN mRNA levels were indeed inversely correlated with miR-30e and stable mir-30e overexpression significantly reduced CRBN mRNA in these cells (MM1S-30e and OPM2-30e). In addition to CRBN, BLIMP1 mRNA and protein levels were also reduced in miR-30e overexpressing cells. In plasma cells, BLIMP1 drives XBP1 expression while supressing c-myc. In MM1S-30e and OPM2-30e (relative to empty vector), and consistent with their reduced BLIMP1 expression, XBP1 mRNA and protein levels were reduced. Furthermore, treatment with lenalidomide (10μM) significantly reduced c-MYC protein levels in MM1S-EV cells after 4 hours while it had no effect on C-MYC expression in MM1S-30e cells.
miR-30e is overexpressed in resistant MM cells and is here shown to regulate cereblon expression, plasma cells differentiation axis (BLIMP1, XBP1) and cell growth (c-MYC).
Neri:Johnson ans Johnson: Research Funding. Bahlis:Johnson and Johnson: Honoraria, Research Funding; Celgene: Honoraria.
Asterisk with author names denotes non-ASH members.