Overexpression of miR-146a promotes cell proliferation and migration in a model of diabetic foot ulcers by regulating the AKAP12 axis

Abstract

In the current study, we aimed to study the effect of miR-146a on proliferation and migration in an in vitro diabetic foot ulcer (DFU) model by targeting A-kinase-anchoring protein 12 (AKAP12). An in vitro DFU model was initially established using HaCaT cells derived from human keratinocytes and induced by advanced glycation end products (AGEs). The effects of overexpression of miR-146a on proliferation and migration ability were analysed. The expression levels of miR-146a and AKAP12 were measured by quantitative real-time polymerase chain reaction (qRT-PCR), and AKAP12, hypoxia-inducible factor-1α (HIF-1α), Wnt3a and β-catenin protein levels were measured by western blotting. The cell proliferation ability was measured by MTT, and the migration ability was analysed by a cell scratch assay. The binding between miR-146a and AKAP12 was identified using a luciferase reporter assay. The results demonstrated that AGEs significantly suppressed cell proliferation and migration, while the expression of miR-146a decreased and the expression of AKAP12 increased. A luciferase reporter assay revealed that miR-146a could directly target AKAP12. Overexpression of miR-146a promoted cell proliferation and migration in an in vitro DFU model and also promoted the expression of HIF-1α, Wnt3a and β-catenin but suppressed the expression of AKAP12. Co-overexpression of miR-146a and AKAP12 reversed the effect of miR-146a on cell proliferation and migration. Our findings revealed that miR-146a directly targeted AKAP12 and promoted cell proliferation and migration in an in vitro DFU model. This study provides a new perspective for the study of miR-146a in the treatment of DFU.

Effect and mechanism of microRNAs on various diabetic wound local cells

“…41 In another in vitro DFU model, the expression of miR-146a was decreased in human keratinocytes, with increased expression of its target A-kinase anchoring protein 12 (AKAP12), thereby inhibiting the proliferation and migration of keratinocytes by blocking HIF-1α/Wnt/β-catenin axis. 42 In addition to affecting the classical repair function of keratinocytes, some miRs abnormally expressed in keratinocytes can also lead to the production of inflammatory responses. The levels of miR-17 to 92 clusters such as miR-19a/b and miR-20a, targeting SHCBP1 and SEMA7A respectively, were reduced in keratinocytes at the edge of human chronic wounds.…”

“…31 MiR-146a was found to be low expressed in both macrophages and keratinocytes, resulting in increased expression of TLR4 and AKAP1, respectively, leading to difficulties in macrophage transformation to M2 phenotype 22 and inhibition of keratinocyte function. 42 In addition, bone marrow stem cells-exosomal vesicles with high expression of miR-146a can improve the function of vascular ECs. 52 In summary, it can be found that miR-146a plays an important role in a variety of cells in the local wound and shows a positive role in promoting wound repair.…”

E2F2 Promotes Wound Healing of Diabetic Foot Ulcer by Regulating CDCA7L Transcription

“…Spontaneously transformed human keratinocyte cell culture (HaCaT) cells [16,17] and HUVECs [14] were purchased from the American Type Culture Collection (Manassas, VA, USA) and incu bated in Dulbecco's Modified Eagle Medium encompassing 10 % fetal bovine serum (Gibco, Grand Island, NY, USA) and 1 % penicil lin/streptomycin at 37 °C with 5 % CO 2 .…”

Natural Biologics Accelerate Healing of Diabetic Foot Ulcers by Regulating Oxidative Stress

“…The adverse immune microenvironment prevented the wound from progressing to the stage of proliferation and remodeling from the stage of inflammation. In the proliferation phase, AGEs significantly inhibited HaCaT cells proliferation and migration by down-regulating the expression of miR-146a and upregulating the expression of an anchoring protein 12 (AKAP12) [65]. Moreover, excessive activation of the AGEs-RAGE pathway disrupts collagen I maturation and prevents its deposition in the ECM, which also further impairs DFU remodeling [66,67].…”