Yonsei Med J.  2017 Jan;58(1):234-240. 10.3349/ymj.2017.58.1.234.

miR-379 Inhibits Cell Proliferation, Invasion, and Migration of Vascular Smooth Muscle Cells by Targeting Insulin-Like Factor-1

Affiliations
  • 1Department of Cardiology, The First Affiliated Hospital of Xi’an Medical University, Xi’an, Shaanxi Province, China. yong_wang0756@sina.com

Abstract

PURPOSE
MicroRNAs are small non-coding RNAs that play important roles in vascular smooth muscle cell (VSMC) function. This study investigated the role of miR-379 on proliferation, invasion, and migration of VSMCs and explored underlying mechanisms thereof.
MATERIALS AND METHODS
MicroRNA, mRNA, and protein levels were determined by quantitative real-time PCR and western blot. The proliferative, invasive, and migratory abilities of VSMCs were measured by CCK-8, invasion, and wound healing assay, respectively. Luciferase reporter assay was used to confirm the target of miR-379.
RESULTS
Platelet-derived growth factor-bb was found to promote cell proliferation and suppress miR-379 expression in VSMCs. Functional assays demonstrated that miR-379 inhibited cell proliferation, cell invasion, and migration. Flow cytometry results further showed that miR-379 induced apoptosis in VSMCs. TargetScan analysis and luciferase report assay confirmed that insulin-like growth factor-1 (IGF-1) 3'UTR is a direct target of miR-379, and mRNA and protein levels of miR-379 and IGF-1 were inversely correlated. Rescue experiments showed that enforced expression of IGF-1 sufficiently overcomes the inhibitory effect of miR-379 on cell proliferation, invasion, and migration in VSMCs.
CONCLUSION
Our results suggest that miR-379 plays an important role in regulating VSMCs proliferation, invasion, and migration by targeting IGF-1.

Keyword

Vascular smooth muscle cells; miR-379; cell proliferation; invasion; migration; IGF-1

MeSH Terms

Apoptosis
Cell Movement/*physiology
Cell Proliferation/*physiology
Humans
Insulin
Insulin-Like Growth Factor I/*physiology
MicroRNAs/*physiology
Muscle, Smooth, Vascular/*cytology
Proto-Oncogene Proteins c-sis/*physiology
RNA, Messenger/metabolism
Real-Time Polymerase Chain Reaction
Sincalide/physiology
Wound Healing/physiology
Insulin
Insulin-Like Growth Factor I
MicroRNAs
Proto-Oncogene Proteins c-sis
RNA, Messenger
Sincalide

Figure

  • Fig. 1 The effect of PDGF-bb on cell proliferation and miR-379 expression in VSMCs. (A) PDGF-bb promotes cell proliferation of VSMCs in a concentration-dependent (10–30 ng/mL) manner as measured by CCK-8 assay. (B) PDGF-bb promotes cell proliferation of VSMCs in a time-dependent (24–72 h) manner as measured by CCK-8 assay. (C) Expression of miR-379 on qRT-PCR is down-regulated after treatment with different concentrations of PDGF-bb. (D) Expression of miR-379 on qRT-PCR is down-regulated after treatment with different durations of PDGF-bb. Data represent the mean values of three independent replicates±SEM; significant differences relative to control group are shown as *p<0.05, **p<0.01, ***p<0.001. PDGF-bb, platelet-derived growth factor-bb; VSMCs, vascular smooth muscle cells; qRT-PCR, quantitative real-time PCR; SEM, standard error of the mean.

  • Fig. 2 The effect of miR-379 on cell proliferation, invasion, migration, and apoptosis of VSMCs. Twenty-four hours after cells were transfected with miR-379 mimics, miR-379 inhibitor, or their respective controls, (A) cell proliferation of VSMCs was measured by CKK-8 assay; (B) cell invasion ability of VSMCs was measured by transwell assay (40×); (C) migration ability of VSMCs was measured by wound healing assay (40×); and (D) cell apoptosis of VSMCs was measured by flow cytometry. Data represent the mean values of three independent replicates±SEM; significant differences relative to control group are shown as *p<0.05, **p<0.01. VSMCs, vascular smooth muscle cells; SEM, standard error of the mean.

  • Fig. 3 IGF-1 is a direct target of miR-379. (A) VSMCs were co-transfected with miR-379 mimics or its control with wild type (wt) or mutant (mut) 3'UTR of IGF-1, and luciferase activity was detected. (B) The mRNA expression levels of IGF-1 in VSMCs were determined by qRT-PCR at 24 h after cells were transfected with miR-379 mimics or its control. (C) Protein expression of IGF-1 in VSMCs was measured by western blot at 24 h after cells were transfected with miR-379 mimics or its control. Data represent the mean values of three independent replicates±SEM; significant differences relative to control group are shown as *p<0.05, **p<0.01. IGF-1, insulin-like growth factor-1; VSMCs, vascular smooth muscle cells; 3'UTR, 3' untranslated region; qRT-PCR, quantitative real-time PCR; SEM, standard error of the mean.

  • Fig. 4 The effect of enforced expression of IGF-1 on cell proliferation, invasion, and migration of VSMCs. Twenty-four hours after cells were co-transfected with miR-379 mimics and pcDNA3.1-IGF-1 or their respective controls, (A) cell proliferation of VSMCs was measured by CKK-8 assay; (B) cell invasion of VSMCs was measured by cell invasion assay; and (C) cell migration of VSMCs was measured by wound healing assay. Data represent the mean values of three independent replicates±SEM; significant differences relative to control group are shown as *p<0.05, **p<0.01. IGF-1, insulin-like growth factor-1; VSMCs, vascular smooth muscle cells; SEM, standard error of the mean.


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