Table 1.
Oligonucleotides for cloning miR-130a, shRNA and 3′UTRs of target genes.
Figure 1.
Characterization of EPCs by confocal microscopy and flow cytometric analysis.
(A) Staining of FITC-UEA-1 (a), DiI-Ac-LDL (b), and dual staining of FITC-UEA-1 and DiI-Ac-LDL (c) were detected using a confocal microscope. (B) A representative FSC/SSC plot. (C) Flow cytometric analysis of the cell surface markers of EPCs (CD34, KDR, CD133, CD14 and CD45). The presented experiment is a typical result obtained from three separate experiments.
Figure 2.
MiR-130a suppressed Runx3 expression.
(A–C) Cells were transfected with a miR-130a inhibitor (anti-miR-130a) or a negative control (blank). (A) Runx3 mRNA level measured by real-time PCR (normalized to β-actin), (B–C) Protein expression of Runx3 by Western blotting and respective densitometric measurement results of Runx3, (D) Luciferase reporter assay. The partially complementary miR-130a-binding site found in the Runx3 3′-UTR (or a mutated binding site) was inserted downstream of a luciferase reporter on the psiCHECK™-2 Vector report plasmid and transfected into EPCs with miR-130a mimic or miR-130a inhibitor. *P<0.05 vs. respective scrambled control (blank) groups. #P<0.05 vs. healthy control group. Similar results were obtained in at least three separate experiments and error bars represent the standard deviation from the mean.
Figure 3.
MiR-130a inhibition decreased EPC proliferation, migration and colony formation, but increased EPC apoptosis.
Cells were transfected with a miR-130a inhibitor (anti-miR-130a) or a negative control (blank) (A) Gene expression of miR-130 by real-time PCR (normalized to U6). (B to F) Functional assays of EPCs: proliferation (B), migration (C), colony formation (D), and apoptosis (E, F) of EPCs. *P<0.05 vs. respective scrambled control (blank) groups. #P<0.05 vs. healthy control group. CFU: colony-forming units. Similar results were obtained in at least three separate experiments and error bars represent the standard deviation from the mean.
Figure 4.
The effect of miR-130 and Runx3 on EPC differentiation.
Expression of CD64 and vWF were analyzed by flow cytometry. (A, B) Cells were transfected with a miR-130 inhibitor or both miR-130 inhibitor and lentiviral Runx3. (C, D) cells were transfected with lentiviral miR-130 or both lentiviral miR-130 and lentiviral vector expressing Runx3 shRNA (Runx3-SH). Lenti-V: cells were tranfected with an empty vector. *P<0.05 vs. respective Lenti-V groups. #P<0.05 vs. healthy control group. Similar results were obtained in at least three separate experiments and error bars represent the standard deviation from the mean.
Figure 5.
Runx3 repression promoted EPC function.
(A–D) Cells were transfected with a lentiviral vector expressing Runx3 shRNA to downregulate Runx3 expression (Runx3-SH) or an empty vector (Lenti-V). (A) Runx3 protein levels by Western blotting and densitometric measurement results of Runx3 protein. (B) EPC proliferation assay. (C) EPC colony formation assay (D) Migration assay. (E) Cells were transfected with a lentiviral vector expressing miR-130a or both miR-130a and Runx3. Tubule formation assay was performed. *P<0.05 vs. respective Lenti-V groups. #P<0.05 vs. healthy control groups for A–D. Similar results were obtained in at least three separate experiments and error bars represent the standard deviation from the mean.
Figure 6.
Runx3 mediated miR-130a’s effect on EPC function and miR-130a upregulated VEGF, p-ERK and Akt1.
(A–E) Cells were transfected with both lentiviral miR-130a and lentiviral Runx3 or lentiviral miR-130a alone. (A) mRNA expression of Runx3 by real-time PCR (normalized to β-actin). (B, C) Protein expression of Runx3 by Western blotting. (D, E) Colony formation and migration capacity of EPCs. * P<0.05 vs. respective empty vector groups (Lenti-V). # P<0.05 vs. lenti-130a group. ** P<0.05 between the same processed healthy and diabetic groups (eg. Lenti-130a control groups VS Lenti-130a diabetes groups ) (F, G) EPCs were transfected with a miR-130a inhibitor or a negative control (Blank), or lentiviral miR-130 or an empty vector (Lenti-V). Protein levels of VEGF, p-ERK and Akt1 were measured by Western blotting. *P<0.05 Lenti-130a vs. Lenti-V group or anti-130a vs. scrambled control (blank) group. VEGF: vascular endothelial growth factor; ERK: extracellular signal-regulated kinase; PI3K: phosphatidylinositol 3′-kinase. The presented experiment is a typical result obtained from three separate experiments.