C. E. Liechty1, J. Hu1, C. Zgheib1, K. W. Liechty1, J. Xu1 1University Of Colorado-Anschutz Medical Campus,Department Of Surgery, Laboratory For Fetal And Regenerative Biology,Aurora, CO, USA
Introduction: The diabetic wound healing impairment has been shown to be multifactorial. A central feature of diabetic wounds is the persistence of chronic inflammation, partly due to the prolonged presence of pro-inflammatory (M1) macrophages in diabetic wounds. Persistence of the M1 macrophage phenotype, and failure to transition to the regenerative or pro-remodeling (M2) macrophage phenotype plays an indispensable role in diabetic wound impairment; however, the mechanism underlying this relationship remains unclear. Recently, microRNAs have also been shown to provide an additional layer of regulation of gene expression. In particular, microRNA-21 (miR-21) is essential for inflammatory response. We hypothesized that miR-21 plays a role in regulating inflammation through promoting M1 macrophage polarization and the production of pro-inflammatory cytokines.
Methods: To test our hypothesis, we treated the mouse macrophage cell line RAW264.7 with LPS (10 pg/ml) and (20 ng/ml) for 24 h to generate M1 macrophages, or IL-4 (20 ng/ml) for 24 h to generate M2 macrophages after overnight serum starvation. MiR-21 overexpression was achieved by transfection of miR-21 mimic. In order to examine the effect of inflammation on miR-21 expression, we treated the mouse macrophage cell line RAW264.7 with LPS; a known inducer of inflammation. Cells were treated with 1, 10, or 100ng/ml LPS for 6 hours or 100ng/ml for 2, 4, 6 hours. Real-time PCR analysis to quantify relative gene expression, using GAPDH or U6 as internal control for mRNA or miRNA expression.
Results: M1 polarized macrophages exhibited an upregulation of miR-21, as well as the M1 and pro-inflammatory markers IL-1b, TNFa, iNos, IL-6, and IL-8. Cells exposed to M2 conditions exhibited an upregulation of M2 markers Mrc1 and Arg1, and no significantly change of miR-21. LPS stimulation of inflammation resulted in upregulation of miR-21, as well as the inflammatory markers TNFa and IL-6 in a dose and time dependent manner. Overexpression of miR-21 in RAW264.7 macrophage cells resulted in an upregulation of miR-21 and also increased expression of the M1 markers IL-1b, TNFa, iNos, and IL-6.
Conclusion: These findings provide the first evidence that miR-21 is involved in the regulation of inflammation and promoting M1 macrophage polarization. Dysregulation of miR-21 in diabetic wounds may explain the abnormal inflammation and persistent M1 macrophage polarization seen in diabetic wounds, and may represent a potential role of miR-21 as a therapeutic target to counteract the impaired wound healing response in diabetic wounds.