![Figure 3. Alterations in MSC miRs in 3D modeling and patient vs normal data, and the resulting changes in MSCs after mimic-induced increased expression of miR-199a. (A) Heat map of the 53 mRNAs identified from nanoString analysis from the 3D model samples that are significantly different between cocultured (Co-culture) and monocultured ND-MSCs (MSCs). Filtering was done on original 800 miRs based on high expression (>25 average counts), significance between myeloma vs normal donor groups (P < .05), and high fc threshold (fc >1.5). (B) Heat map of 41 miRs identified from nanoString analysis that are significantly different between patient samples (myeloma [MM] and normal donor [ND] sample MSCs). Filtering was done on original 800 miRs based on high expression (>25 average counts), with significance between myeloma vs normal donor groups (P < .05), and high fc threshold (fc >1.5). (C) Alizarin Red staining quantification of mineralization produced by MSCs transfected with negative control mimic or miR-199a-5p mimic after 10 days in osteogenic no-dexamethasone media. Data plotted as mean ±SEM, n ≥3 different donors. (D) Alizarin Red staining representative images showing mineralization of MM-MSCs in 6-well plates transfected with negative control mimics or miR-199a-5p mimics to increase miR-199a-5p expression after 10 days in osteogenic no-dexamethasone media. Images are representative of n ≥3 different donors. The scale bars represent 200µm (original magnification ×4), 100 µm (×10), 50 µm (×20), and 20 µm (×40). (E) MM-MSCs transfected to increase expression of miR-199a-3p (E) or 199a-5p (F) demonstrate increased expression of osteogenic markers after 10 days of culture, measured by q-RT-PCR, gene expression normalized to negative control (Control) for each gene. ALPL, alkaline phosphatase; BSP, integrin-binding sialoprotein; Col1a1, collagen type I α 1; OP, osteopontin; OC, osteocalcin; RUNX2, runt-related transcription factor 2. Data plotted as mean ± SEM and analyzed with 1-way ANOVA and a post hoc Fisher least significance difference test for multiple comparisons (each gene vs negative control). Day 10 after transfection with miR mimics or controls and grown in osteogenic-no dexamethasone medium. n ≥ 3 different donors, **P < .05, **P < .01.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/124/22/10.1182_blood-2014-02-558007/4/3250f3.jpeg?Expires=1735507742&Signature=2CMasUdJHlLBH4p4okrGKWT2t-2YZphG4JTEU7fn~MqrTMqmo14NbGPjKq9D-VWtmpDiVcwRVb~3zoXuCOs6FGlYslOl9zbFhfPDRhGA7XIfKQM68YtpvZWfp2JRew5h7QIn1IDPyQwGOdz7FnIefKQGx8OBvjWozUn0pph20LlkX6IxJV2hJBesl1D0Eyf3jsLfIulfX-f3irPNYmgpJeoyiad3pb0QfSjA6Vv~LbJS9qAz1Am4J7qCIMF68I4BN9evWMC4wHha3mzrotWwksJnkpOGM-siixf9I1haDC0czA4VSrL9YBYY9xZXuHHt-PKZfVvjJPdF1ZrCAWCBbg__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Alterations in MSC miRs in 3D modeling and patient vs normal data, and the resulting changes in MSCs after mimic-induced increased expression of miR-199a. (A) Heat map of the 53 mRNAs identified from nanoString analysis from the 3D model samples that are significantly different between cocultured (Co-culture) and monocultured ND-MSCs (MSCs). Filtering was done on original 800 miRs based on high expression (>25 average counts), significance between myeloma vs normal donor groups (P < .05), and high fc threshold (fc >1.5). (B) Heat map of 41 miRs identified from nanoString analysis that are significantly different between patient samples (myeloma [MM] and normal donor [ND] sample MSCs). Filtering was done on original 800 miRs based on high expression (>25 average counts), with significance between myeloma vs normal donor groups (P < .05), and high fc threshold (fc >1.5). (C) Alizarin Red staining quantification of mineralization produced by MSCs transfected with negative control mimic or miR-199a-5p mimic after 10 days in osteogenic no-dexamethasone media. Data plotted as mean ±SEM, n ≥3 different donors. (D) Alizarin Red staining representative images showing mineralization of MM-MSCs in 6-well plates transfected with negative control mimics or miR-199a-5p mimics to increase miR-199a-5p expression after 10 days in osteogenic no-dexamethasone media. Images are representative of n ≥3 different donors. The scale bars represent 200µm (original magnification ×4), 100 µm (×10), 50 µm (×20), and 20 µm (×40). (E) MM-MSCs transfected to increase expression of miR-199a-3p (E) or 199a-5p (F) demonstrate increased expression of osteogenic markers after 10 days of culture, measured by q-RT-PCR, gene expression normalized to negative control (Control) for each gene. ALPL, alkaline phosphatase; BSP, integrin-binding sialoprotein; Col1a1, collagen type I α 1; OP, osteopontin; OC, osteocalcin; RUNX2, runt-related transcription factor 2. Data plotted as mean ± SEM and analyzed with 1-way ANOVA and a post hoc Fisher least significance difference test for multiple comparisons (each gene vs negative control). Day 10 after transfection with miR mimics or controls and grown in osteogenic-no dexamethasone medium. n ≥ 3 different donors, **P < .05, **P < .01.
