Integrative analysis of miRNA and mRNA paired expression profiling of primary fibroblast derived from diabetic foot ulcers reveals multiple impaired cellular functions.
Diabetic foot ulcers (DFUs) are one of the major complications of diabetes. Its molecular pathology remains poorly understood, impeding the development of effective treatments. Although it has been established that multiple cell types, including fibroblasts, keratinocytes, macrophages, and endothelial cells, all contribute to inhibition of healing, less is known regarding contributions of individual cell type. Thus, we generated primary fibroblasts from nonhealing DFUs and evaluated their cellular and molecular properties in comparison to nondiabetic foot fibroblasts (NFFs). Specifically, we analyzed both micro-RNA and mRNA expression profiles of primary DFU fibroblasts. Paired genomic analyses identified a total of 331 reciprocal miRNA-mRNA pairs including 21 miRNAs (FC > 2.0) along with 239 predicted target genes (FC > 1.5) that are significantly and differentially expressed. Of these, we focused on three miRNAs (miR-21-5p, miR-34a-5p, miR-145-5p) that were induced in DFU fibroblasts as most differentially regulated. The involvement of these microRNAs in wound healing was investigated by testing the expression of their downstream targets as well as by quantifying cellular behaviors in prospectively collected and generated cell lines from 15 patients (seven DFUF and eight NFF samples). We found large number of downstream targets of miR-21-5p, miR-34a-5p, miR-145-5p to be coordinately regulated in mRNA profiles, which was confirmed by quantitative real-time PCR. Pathway analysis on paired miRNA-mRNA profiles predicted inhibition of cell movement and cell proliferation, as well as activation of cell differentiation and senescence in DFU fibroblasts, which was confirmed by cellular assays. We concluded that induction of miR-21-5p, miR-34a-5p, miR-145-5p in DFU dermal fibroblasts plays an important role in impairing multiple cellular functions, thus contributing to overall inhibition of healing in DFUs.
Department of Dermatology & Cutaneous Surgery, University of Miami Miller School of Medicine (UMMSOM), Wound Healing and Regenerative Medicine Research Program.
Liang
L
Liang
Department of Dermatology & Cutaneous Surgery, University of Miami Miller School of Medicine (UMMSOM), Wound Healing and Regenerative Medicine Research Program.
Rivka C
RC
Stone
Department of Dermatology & Cutaneous Surgery, University of Miami Miller School of Medicine (UMMSOM), Wound Healing and Regenerative Medicine Research Program.|Wound Healing Clinical Research Program, UM Health System, Miami, Florida.
Olivera
O
Stojadinovic
Department of Dermatology & Cutaneous Surgery, University of Miami Miller School of Medicine (UMMSOM), Wound Healing and Regenerative Medicine Research Program.|Human Genomics and Genetics Graduate Program, Tufts University, Boston, Massachusetts.
Horacio
H
Ramirez
Department of Dermatology & Cutaneous Surgery, University of Miami Miller School of Medicine (UMMSOM), Wound Healing and Regenerative Medicine Research Program.
Irena
I
Pastar
Department of Cell, Molecular, and Developmental Biology, Sackler School of Graduate Biomedical Sciences, Tufts University, Boston, Massachusetts.|Department of Oral and Maxillofacial Pathology, School of Dentistry, School of Medicine, School of Engineering, Tufts University, Boston, Massachusetts.
Anna G
AG
Maione
John P. Hussman Institute for Human Genomics, University of Miami Miller School of Medicine (UMMSOM), Miami, Florida.
Avi
A
Smith
John P. Hussman Institute for Human Genomics, University of Miami Miller School of Medicine (UMMSOM), Miami, Florida.
Vanessa
V
Yanez
Beth Israel Deaconess Medical Center, Boston, Massachusetts.
Aristides
A
Veves
Department of Dermatology & Cutaneous Surgery, University of Miami Miller School of Medicine (UMMSOM), Wound Healing and Regenerative Medicine Research Program.|Wound Healing Clinical Research Program, UM Health System, Miami, Florida.
Robert S
RS
Kirsner
Department of Cell, Molecular, and Developmental Biology, Sackler School of Graduate Biomedical Sciences, Tufts University, Boston, Massachusetts.|Department of Oral and Maxillofacial Pathology, School of Dentistry, School of Medicine, School of Engineering, Tufts University, Boston, Massachusetts.
Jonathan A
JA
Garlick
Department of Dermatology & Cutaneous Surgery, University of Miami Miller School of Medicine (UMMSOM), Wound Healing and Regenerative Medicine Research Program.|Wound Healing Clinical Research Program, UM Health System, Miami, Florida.|Human Genomics and Genetics Graduate Program, Tufts University, Boston, Massachusetts.|John P. Hussman Institute for Human Genomics, University of Miami Miller School of Medicine (UMMSOM), Miami, Florida.
Marjana
M
Tomic-Canic
D015153:Blotting, Western; D002454:Cell Differentiation; D016922:Cellular Senescence; D017719:Diabetic Foot / Q000235:genetics* / Q000473:pathology*; D005347:Fibroblasts / Q000378:metabolism* / Q000473:pathology*; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D006801:Humans; D007150:Immunohistochemistry; D035683:MicroRNAs / Q000378:metabolism*; D046228:Microarray Analysis; D012333:RNA, Messenger / Q000378:metabolism*; D015398:Signal Transduction; D014945:Wound Healing
D035683:MicroRNAs; D012333:RNA, Messenger