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

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

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

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