AMF siRNA treatment of keloid through inhibition signaling pathway of RhoA/ROCK1

Abstract

A keloid (KD) is a benign dermal fibrotic tumor. Treatment of KDs is challenging and the recurrence rate is high; thus, there is an unmet need to explore new target sites and new treatment methods. As a tumor-associated cytokine, autocrine motility factor (AMF) can effectively stimulate the random and directional movement of cells. We first found that AMF was overexpressed in keloid fibroblasts (KFs) and the proliferation and migration of KFs were promoted by AMF stimulation. After treatment with Y-27632, RhoA kinase inhibitor, the proliferation and migration capacity of KFs declined significantly, and type I collagen protein, active RhoA and ROCK1 also were downregulated. In addition, a KD transplantation model was established under the skin of nude mice, with KD intramural injection AMF siRNA, we found that the weight of the KD was smaller than in the control group (P < 0.05), KD tissue sections stained by HE and Masson showed that fibers became loose and the blood vessels were visibly reduced. In conclusion, AMF siRNA is expected to be a novel strategy to treat KD by inhibiting signaling pathway of RhoA/ROCK1.

Keywords: Autocrine motility factor; Keloid; RhoA; SIRNA; Treatment.

Figure 1

The expression of AMF. The tissue immunofluorescence staining in the PBS negative control group (A), normal epidermal tissue (B), hypertrophic scar (C) and KD (D). Scale bars: 500 μm. The mRNA (E) and protein (F) expression of NFs, HFs, and KFs. *P < 0.05.

Figure 2

The effect of the stimulation with different concentrations of AMF at different times on KFs. The absorbance value of KFs after stimulation with different concentration (A) and different time points (B), the migration of KFs (C), and the expression of the protein of collagen I, total RhoA, active RhoA, and ROCK1 (D). *P < 0.05, **P < 0.01.

Figure 3

Effect of RhoA/ROCK1 signaling pathway on the biological behavior of KFs challenged with Y-27632. The absorbance value (A), the number of migration KFs (B), and the expression of collagen I, active RhoA, total RhoA, and ROCK1 (C). **P < 0.01.

Figure 4

The therapeutic effect of AMF siRNA on KD in nude mice. KD-bearing mice and KD specimens (A), the weight of keloid (B), HE staining, Scale bars: 25 μm (C) and Masson staining, Scale bars: 50 μm (D) at the end of different treatments. *P < 0.05.