Long noncoding RNA GAS5 accelerates diabetic wound healing and promotes lymphangiogenesis via miR-217/Prox1 axis.

BACKGROUND: Diabetes is usually the leading cause of chronic non-healing wounds. LncRNA-GAS5 has been verified to be involved in the regulation of diabetes or high glucose (HG)-stimulated cells. However, its regulatory roles in diabetic wound healing need further investigation. METHOD: GAS5, miR-217 and Prox1 were identified by qRT-PCR. MTT, flow cytometry assay, wound-healing assay and tube formation were used to analyze cell viability, apoptosis, migration and tube formation capacity. Western blotting was carried out to detect the protein expression of c-Myc, CyclinD1, CDK4, Bcl-2, Prox1, VEGFR-3 and LYVE-1. Bioinformatics and luciferase assay were performed to predict and validate the binding sites of miR-217 on GAS5 and Prox1. Immunofluorescence staining detected the expression and distribution of Prox1. The wound healing rate was also assessed by setting up the diabetic mouse model. H&E staining assessed the distribution of inflammatory cells and fibroblasts in the wound tissues. RESULTS: GAS5 was significantly down-regulated whereas miR-217 was obviously up-regulated in diabetic skin, HG-induced lymphatic endothelial cells (LECs) and diabetic mouse model. GAS5 sponged miR-217 to up-regulate Prox1. GAS5 overexpression or miR-217 inhibition rescued the impairments of cell viability, migration and lymphatic vessel formation and the facilitation of apoptosis of LECs caused by HG. Similar impacts were observed on the protein level of VEGFR-3, LYVE-1, and Prox1. GAS5 promoted wound healing and lymphangiogenesis in the diabetic mouse model. CONCLUSION: GAS5 sponged miR-217 to up-regulate Prox1 and promote lymphangiogenesis and diabetic wound healing. This might provide novel therapeutic strategy to improve the efficacy of diabetic wound healing.

Long noncoding RNA-antisense noncoding RNA in the INK4 locus accelerates wound healing in diabetes by promoting lymphangiogenesis via regulating miR-181a/Prox1 axis.

BACKGROUND: Slow lymphangiogenesis is one crucial reason for the impaired wound healing process in diabetes. Accumulative evidence showed that long noncoding RNA-antisense noncoding RNA in the INK4 locus (ANRIL) could influence lymphangiogenesis. Besides, miR-181a has been reported to regulate Prox1 that is essential for lymphangiogenesis. However, the relationship between ANRIL and miR-181a as well as the definitive function of ANRIL in lymphangiogenesis is not clear. METHODS: The diabetic mouse model was set up to assess the wound healing rate in vivo. Quantitative real-time polymerase chain reaction was performed to measure the expressions of ANRIL, miR-181a, and Prox1. Western blot analysis was used to assess the expressions of vascular endothelial growth factor receptor-3, lymphatic vessel hyaluronan receptor-1, Prox1, and epithelial-mesenchymal transition (EMT)-related proteins. Flow cytometry was used to assess the cell apoptosis. Wound healing assay was used to determine the effect of ANRIL on cell migration. Tube-formation assay and immunofluorescence staining were performed to determine tube-formation capacity of human dermal lymphatic endothelial cells (LECs). RESULTS: ANRIL and Prox1 were downregulated, whereas miR-181a was upregulated in the diabetic wound healing mouse model and high glucose (HG)-induced LECs. The wound healing rate and EMT were inhibited during the diabetic wound healing process. Dual-luciferase assay proved that miR-181a could bind Prox1 to repress its expression, whereas ANRIL could sponge miR-181a to recover Prox1 expression. Overexpression of ANRIL or inhibition of miR-181a rescued the impairments of survival, migration, EMT formation, and tube formation of LECs caused by HG. CONCLUSION: ANRIL could promote lymphangiogenesis during the diabetic wound healing process via sponging miR-181a to enhance Prox1 expression, which might help design new therapy to improve the wound healing efficacy for diabetes.