Global Rnaseq/Proteomic-Phoshoproteomic Analysis Unveil Mir-21 As a Central Player in Driving Th17 Mediated Bone Disease in MM

Background Bone disease (BD) is a hallmark of multiple myeloma (MM) and is characterized by severe skeleton damage, reduced quality of life and overall survival (1-2). Several findings indicated that IL-17 producing CD4+ T cells (Th17) play a central role in triggering MMBD and support MM cell growth mainly by IL-17 production. There is compelling evidence that miR-21 is a central player in Th17 effector functions. Our preliminary data have shown that miR-21 is highly upregulated in MM-Th17 isolated from patients with active BD as compared to MM with no active BD and controls. We found that inhibition of miR-21 in naive T cells (miR-21i-T cells) impaired differentiation towards Th17 in vitro, by reducing interleukin (IL)-17, IL-22, RANKL and RORC, leading to abrogation of osteoclast (OCL) bone resorption. Aims Based on these premises, we sought to explore miR-21 related underlying molecular networks that support pathogenic Th17 differentiation and function. As miRNAs may exert direct and indirect effects on gene expression and at post-transcriptional level, we performed a global head-to-head comparison by RNA-seq and proteomic -phosphoproteomic analysis on miR-21i-Th17. Then, we recapitulated and validated our findings in NOD/SCID gNULL mice, injected intratibially with miR-21i-T cells and MM cells. Methods RNAseq and proteomic/phosphoproteomic assays have been performed on in vitro differentiated Th17 cells originated from scramble control (SC) or miR-21i transfected naïve T cells (SC-Th17 and miR-21i-Th17 respectively) from 3 healthy donors through MARS-seq protocol adapted for bulk RNA and proteome/phosphoproteome analysis . Data have been analyzed through R by using different packages including limma, DESEQ2 and pheatmap. To perfom global proteome/phosphoproteome analysis, we conducted a mass spectrometry study of phosphopeptides protein extract from SC-Th17 and miR-21i-Th17, enriched using SCX-IMAC/TiO2. High-resolution LC-Ms/MS data were processed using Proteome Discoverer software Results In the presence of miR-21i, we found 109 upregulated and 22 downregulated proteins in the global proteome analysis of Th17 cells, while 90 and 18 phosphoproteins were up and down modulated, respectively. Paired analysis showed that 46 proteins are modulated in expression but not in phosphorylation, 23 proteins are modulated in phosphorylation but not in expression, while 85 proteins are modulated in both conditions. These data suggest that selective miRNA modulatio