Inhibition of miR-135b has a therapeutic effect to restore the impaired osteogenic potential of MM-hMSCs.

<p>(A) Transfection of miR-135b inhibitor (50nM) in osteogenic induction medium (OM) for 3 days significantly increases ALP activity for MM-hMSCs. n=4. * <i>p</i><0.05; NS, not significant, compared to mock group. (B) MM-hMSCs are cultured in OM supplemented with miR-135b inhibitor (50nM), miRNA inhibitor negative control or transfection reagent only for one week. Bone formation markers (<i>BSP</i>, <i>COLA1</i> and <i>OPN</i>) show a significant increase in miR-135b knockdown MM-hMSCs by quantitative real time PCR. n=3. * <i>p</i><0.05; NS, not significant, compared to mock group. (C) ALP staining confirms that loss of miR-135b results in an increase in MM-hMSCs osteogenic differentiation. One representative result of three is shown. (D) Alizarin Red S staining also confirms that MM-hMSCs transfected with miR-135b inhibitor demonstrated an increased calcium deposit. Original magnification, ×100. Scale bar, 200μm. One representative result of three is shown. (E) Upregulation of miR-135b in ND-hMSCs is induced by coculture of MM cells RPMI8226 and U266, and gradually returns to normal level after removal of MM cells. n=3. * and # indicate <i>p</i><0.05, compared to day 0 for U266 and RPMI8226 coculture, respectively. ND-hMSCs without MM cell coculture were used to exclude the possible influence of cell confluence on miR-135b expression.