Figure 6.

The effect of a miR-7977 mimic on MSCs. (A) The reduction in JAG1 was analyzed by western blotting analysis. Control, 5 nM negative control siRNA; miR-7977, 5 nM miR-7977 mimic; Control protector, 250 nM negative control miScript Target Protector; JAG1 protector, 250 nM Target Protector for JAG1. (B) The reduction in PCBP1 was analyzed by western blotting analysis. PCBP1 protector, Target Protector for PCBP1. Direct interaction between miR-7977 and target genes was evaluated by luciferase assay using pLuc-JAG1 (C) and pLuc-PCBP1 (D). Data are presented as the ratio of the normalized value to the light emission observed in the cells transduced with the control vector (pLuc-Cont). pMIR-Cont or pMIR-7977 was cotransfected with pLuc-JAG1 or pLuc-PCBP1 into MSCs 48 hrs before analysis (Online Supplementary Methods). Data shown are from one representative experiment of three showing similar results, each done in quadruplicate. Results are expressed as means ± SD. *P<0.05, pMIR-Cont with pLuc-Cont vs. pMIR-7977 with pLuc-JAG1. **P<0.01, pMIR-Cont with pLuc-Cont vs. miR-7977 with pLuc-PCBP1. (E) The expression of JAG1 mRNA in MSCs after transfer of 5 nM miR7977 mimic and 250 nM PCBP1 protector was examined by qRT-PCR. *P<0.05, HV-derived MSCs transduced with negative control vs. HV-derived MSCs transduced with miR-7977 mimic. ^†P<0.05 MSCs transfected with miR-7977 mimic vs. MSCs cotransfected with miR-7977 mimic and PCBP1 protector (Student’s t-test). Y-axis indicates fold change relative to the control or control + GW4869 (=1.0) after normalization to 18S expression level in MSCs. Data shown are from one representative experiment of three showing similar results, each done in quadruplicate. (F) The recovery of SCF and ANGPT1 expression after PCBP1 protector transfer into MSCs was analyzed by western blotting analysis.