MicroRNA signature in diabetic wound healing: promotive role of miR-21 in fibroblast migration

Abstract

A major complication of diabetes mellitus is the disruption of normal wound repair process, characterised by insufficient production of growth factors. A molecular genetic approach wherein resident cells synthesise and deliver the growth factors to the wound site would be a powerful therapeutic strategy to treat diabetic wounds. One such molecular approach could be the application of microRNAs (miRNAs). This study reports differential expression of miRNAs related to cell development and differentiation, during wound healing in diabetic mice. Comparison of skin tissue from normal and diabetic mice showed that 14 miRNAs were differentially expressed in diabetic skin; miR-146b and miR-21 were the most noteworthy. Expression pattern of these miRNAs was also altered during healing of diabetic wounds. A subset of miRNAs (miR-20b, miR-10a, miR-10b, miR-96, miR-128, miR-452 and miR-541) exhibited similar basal levels in normal and diabetic skins, but displayed dysregulation during healing of diabetic wounds. Amongst the miRNAs studied, miR-21 showed a distinct signature with increased expression in diabetic skin but decreased expression during diabetic wound healing. We analysed the role of miR-21 in fibroblast migration, because migration of fibroblasts into the wound area is an important landmark facilitating secretion of growth factors and migration of other cell types into the wound, thus enhancing the healing process. Using gain-of and loss-of function approaches, we show that miR-21 is involved in fibroblast migration. Our preliminary studies implicate an important role for miRNAs in the pathogenesis of diabetic wounds.

Figure 1

Dermal wound healing in normal (ICR) versus diabetic (KKAY) mice: Full‐thickness punch wounds were created on the backs of the mice as explained in Materials and Methods section and the wounds were monitored over a period of 8 days. The middle panels show a closer view of the wounds. Images shown are representative of three independent experiments.

Figure 2

Signature of miRNAs during healing of normal and diabetic wounds: Real‐time polymerase chain reaction (RT² PCR) array was performed on unwounded skin tissues obtained from normal and diabetic mice. miRNA expression in tissues of diabetic mice was expressed as fold increase in comparison to tissues of normal mice (A). Wound tissues were obtained on days 0, 4 and 8 post wounding and analysed for differential expression. Panel (B) represents miRNAs that expressed <20‐fold change in diabetic wounds in comparison to normal wounds; panel (C) represents miRNAs that expressed >20‐fold change in diabetic wounds versus normal wounds. Panel (D) represents the subset of miRNAs that were similarly expressed in skin tissues of normal and diabetic mice but were dysregulated during healing of diabetic wounds. Data represent average of three independent experiments.

Figure 3

Expression pattern of miR‐21 during healing of normal and diabetic wounds: Wound tissues were obtained on days 0, 4 and 8 post wounding and analysed for miR‐21 expression. Expression level in normal wounds on day 0 was used as a standard to evaluate the expression pattern during wound healing. Data represent average of three independent experiments.

Figure 4

miR‐21 enhances migration of fibroblasts towards the wound: Human skin fibroblasts were transfected with precursor (Pre miR‐21) or antagonist (Anti miR‐21) to miR‐21 and subjected to in vitro wound healing assay as described in Materials and Methods section. Black line denotes the wound edge created during the start of the assay. Digital images were obtained at 0 and 18 hours post wounding. Images shown are representative of three independent experiments.