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Background: Keratinocyte migration is essential for skin wound healing and recent studies demonstrated that microRNAs (miRNAs) are involved in the differentiation, migration and apoptosis in keratinocytes. However, the function of miR-26a in wound healing remains to be largely explored. Methods: Northern blot and quantitative reverse transcriptase PCR (qRT-PCR) were used to detect the miR-26a expression and Western blot was used to detect integrin α-5 (ITGA5), phosphatidylinositol-3-kinase (PI3K), p-PI3K, protein kinase B (AKT) and p-AKT protein expression in immortalized human keratinocyte cell line HaCaT and normal human epidermal keratinocytes (NHEK) after 2 ng/ml transforming growth factor-β1 (TGF-β1) treatment for 0, 6, 12 and 24 h. Transwell assay and Wound healing assay were introduced to measure the cell migration of HaCaT cells. TargetScan online database, luciferase reporter assay and RNA immunoprecipitation (RIP) were employed to confirm the relationship between miR-26a and ITGA5. Results: The RNA expression of miR-26a was down-regulated and ITGA5 protein expression was up-regulated by TGF-β1 treatment in HaCaT and NHEK cells in a time-dependent manner. MiR-26a overexpression inhibited the migration of HaCaT cells induced by TGF-β1 while miR-26a inhibitor enhanced the migration. ITGA5 was a downstream target mRNA and regulated by miR-26a. ITGA5 overexpression reversed the inhibitory effect of miR-26a on migration in HaCaT, while ITGA5 knockdown attenuated the stimulative effect of miR-26a inhibitor in HaCaT via PI3K/AKT signaling pathway. Conclusion: MiR-26a overexpression inhibited TGF-β1 induced HaCaT cells migration via down-regulating ITGA5 through activating the PI3K/AKT signaling pathway.

Purpose: Plaque stability is governed by it's collagen content and VSMC phenotype1. RGS5, a GPCR modulator, has been shown to be associated with plaque stability3. RGS5 may moderate VSMC/endothelial cell interactions4. We examined if RGS5 expression is associated with plaque stability and detailed its role in VSMC/HUVEC behavior in vitro. Methods: With IRB permission, samples from 91 patients undergoing carotid thrombendarterectomy were collected and RNA was extracted. Target-PCRs for RGS5 were normalized for PCR efficiency and 3 housekeeping genes (GAPDH, ATPsynthase, Tubulin) using qBase software. RGS5 was downregulated in human VSMC or HUVEC by siRNA. After 24h, cells were incubated with mytomicin-C for 1 hour, and the confluent monolayer was wounded. Wound closure percentage after 20h, of scrambled vs. RGS5 siRNA transfected VSMCs was measured (ImageJ). HUVECs conditioned medium was used to perform VSMC scratch assays. RGS5 expression in resident vs. migrated VSMCs was assessed by collecting the cells that had migrated off a confluent coverslip. RNA was extracted for qRT-PCR. RGS5 expression was normalized by HPRT using the Δ(ΔCT) method. Scrambeled and RGS5 siRNA transfected HUVECs were cocultured with VSMCs to produce spheroids in a 96 well plate (1:1 ratio, 3000 cell/well). Spheroids structure was scored 24 hours later for HUVEC coverage, spheroid shape and size. Results: RGS5-RNA was increased in stable compared to unstable plaques (3.1±0.5 vs 1.6±0.3 CNRQ, n=91, p