Upregulation of miR-135b Is Involved in the Impaired Osteogenic Differentiation of Mesenchymal Stem Cells Derived from Multiple Myeloma Patients

<div><p>Previous studies have demonstrated that mesenchymal stem cells from multiple myeloma (MM) patients (MM-hMSCs) display a distinctive gene expression profile, an enhanced production of cytokines and an impaired osteogenic differentiation ability compared to normal donors (ND-hMSCs). However, the underlying molecular mechanisms are unclear. In the present study, we observed that MM-hMSCs exhibited an abnormal upregulation of miR-135b, showing meanwhile an impaired osteogenic differentiation and a decrease of SMAD5 expression, which is the target of miR-135b involved in osteogenesis. By gain and loss of function studies we confirmed that miR-135b negatively regulated hMSCs osteogenesis. We also found that MM cell-produced factors stimulated ND-hMSCs to upregulate the expression of miR-135b. Importantly, treatment with a miR-135b inhibitor promoted osteogenic differentiation in MM-hMSCs. Finally, we observed that MM cell-derived soluble factors could induce an upregulation of miR-135b expression in ND-hMSCs in an indirect coculture system and the miR-135b expression turned to normal level after the removal of MM cells. Collectively, we provide evidence that miR-135b is involved in the impaired osteogenic differentiation of MSCs derived from MM patients and might therefore be a promising target for controlling bone disease. </p> </div

miR-135b negatively regulates SMAD5 expression.

<p>(A) Transfection with miR-135b inhibitor or mimic leads to a decrease or an increase of miR-135b expression in hMSCs. One of three independent experiments is shown. (B) miR-135b is confirmed to target SMAD5 in hMSCs. Transfection with miR-135b inhibitor or mimic leads to an increase or a decrease of SMAD5 expression, respectively, by Western blot in hMSCs. One of three independent experiments is shown. (C) HEK 293 cells were cotransfected with the luciferase reporters carrying wild-type or mutated SMAD5 3’UTR, and 50 nM miR-135b mimic or negative control (miR-C) for 48 h. The luminescence of Renilla luciferase was normalized to that of firefly luciferase, and the relative luminescence units was plotted. Normalized data are shown as mean±SD. n=3. ***, p<0.001; NS, not significant. (D) MM-hMSCs with upregulated miR-135b expression have a lower SMAD5 expression as shown by Western blot analysis. Left panels show the blots; right panels show densitometric analysis using ImageJ software. *<i>p</i><0.05.</p

miR-135b expression is higher in MM BM-derived hMSCs showing impaired osteogenic differentiation potential.

<p>(A) MM-hMSCs show a reduced ALP activity, a marker for osteogenic differentiation. ND-hMSCs (n=7) and MM-hMSCs (n=12) are cultured in osteogenic medium (OM) for 72 hours and the ALP activity is measured quantitatively by the alkaline phosphatase yellow (pNPP) liquid substrate system for ELISA (Sigma-Aldrich, Bornem, Belgium). ** <i>p</i><0.01 (B) miR-135b expression is significantly upregulated in MM-hMSCs compared to ND-hMSCs by quantitative real time PCR. The miR-135b expression of all hMSCs samples is normalized to the miR-135b expression of U266 MM cells. * <i>p</i><0.05 (C) miR135b expression is inversely correlated with the ALP activity in MM-hMSCs. The relative expression of miR-135b in hMSCs derived from 12 MM patients was plotted versus their ALP activity.</p

MM-hMSCs exhibit a different miR-135b expression during osteogenic differentiation compared to ND-hMSCs.

<p>(A) The osteogenic differentiation of ND-hMSCs and MM-hMSCs gradually progressed when exposed to osteogenic induction medium (OM) <i>in </i><i>vitro</i> as shown by qualitative ALP staining. However, the ALP increase in MM-hMSCs is lower compared to ND-hMSCs. (B) miR-135b relative expression, as detected by quantitative real time PCR, decreases during osteogenesis significantly in both ND-hMSCs and MM-hMSCs. There is a delay of miR-135b decrease in MM-hMSCs. n=5/group. * and # indicate <i>p</i><0.05, compared to day 0 for ND-hMSCs and MM-hMSCs, respectively. All values are normalized to day 0. (C) MM-hMSCs with impaired osteogenic differentiation show a less increasing level of SMAD5 during osteogenic differentiation compared to ND-hMSCs. One representative result of three is shown.</p

Distribution of <i>MYD88</i> L265P allele fractions in samples from patients with familial Waldenström Macroglobulinemia prior to or after immortalization by EBV, and in lymphoblastoid lines derived from patients with lung cancer.

<p>Distribution of <i>MYD88</i> L265P allele fractions in samples from patients with familial Waldenström Macroglobulinemia prior to or after immortalization by EBV, and in lymphoblastoid lines derived from patients with lung cancer.</p

INSURE: A pooled analysis of ixazomib-lenalidomide-dexamethasone for relapsed/refractory myeloma in routine practice - supplementary material

Supplementary methods, tables and figures </p

Identification and expression of miRNA candidates in MM.

<p>(A) Effect of 5-aza-CdR on the expression of miR-152, -10b-5p and miR-34c-3p in six MM cell lines by real-time PCR. Experiments were performed in triplicate and RNU6B was used as the internal control. Data were shown as means ± SD. ^*<i>P</i><0.05 compared with cells without 5-aza-CdR. (B) Expression levels of miR-152 and miR-10b-5p in MM and healthy donor in GSE16558.</p

Methylation of miR-152, -10b-5p and miR-34c-3p in MM cells.

<p>(A) Schematic illustration of the percentage of C + G nucleotides (CG%) and the density of CpG olinucleotides were shown for a region spanning 1000 bp upstream and 500 bp downstream of miR-152, miR-10b-5p and miR-34c-3p, respectively. Specific primers for these CpG islands were designed (arrows) and used to amplify these DNA fragments in MM cell lines. The CpG island was depicted, and each vertical bar illustrated a single CpG. (B) Representative MSP results of the three miRNAs methylation inMM1S, RPMI 8266, OPM2, U266, IM9 and H929 cell lines. M: methylated primers; U: unmehtylated primers. PC: positive control. (C) Bisulfite sequencing analysis showed relative methylation frequencies of miR-152, -10b-5p and miR-34c-3p in six MM cell lines. Eight single clones for each sample were selected and T7 primers were used for sequencing. (D) Bisulfite sequencing analysis showed methylation frequencies of miR-152 and miR-34c-3p in H929 and IM9, and miR-10b-5p in H929 and MM1S, treated with or without 5′-aza-CdR (5 µM) for 4 days. Black and white circle represented methylated and unmethylated CpG, respectively. (E) Representative sequencing results showed that the cytosine (C) residues of CpG dinucleotides were converted into thymidine (T).</p

Effect of miR-152 and miR-10b-5p on the expression of putative targets.

<p>(A) GSEA established that predicated targets of miR-152 and miR-10b-5p was positively correlated with MM, and negatively correlated with HD. NES: normalized enrichment score; FDR: false discovery rate. (B) The expression of predicted targets of miR-152 (DNMT1 and E2F3) and miR-10b-5p (E2F3, BTRC and MYCBP) in miR-152-, -10b-5p- or NC-transfected MM cells by real-time PCR with normalization to the reference 18S expression. ^*<i>P</i><0.05 compared with NC-transfected cells. (C) The expression of DNMT1, E2F3, BTRC and MYCBP in healthy donor and MM (GSE5900 and GSE2658).</p

miR-152, -10b-5p and miR-34c-3p modulated proliferation and apoptosis of MM cells.

<p>Since the three miRNAs have different methylation frequencies and expression levels in MM cell lines, H929 cells were transfected with miR-152, -10b-5p and -34c-3p mirVana miRNA respectively; IM9 cells were transfected with miR-152 and -34c-3p mirVana miRNA respectively; MM1S cells were used to overexpress miR-10b-5p. All of the cells transfected with negative control mimics used as control cells. (A) The indicated transfected cells were cultured in presence or absence with primary BMSCs for 72 hours, following by [H]³ thymidine uptake assay. NC-transfected cells in absence with BMSCs were defined as 1.0 and regarded as the control group. ^*<i>P</i><0.05. (B) MM cells (NC-, miR-152-, -10b-5p-, or miR-34c-3p-transfected) were harvested at 48 or 72 hours after transfection. Whole cell lysates were subjected to western blotting using anti-PARP, -Caspase 9, -Caspase 3 and -α-tubulin antibodies.</p