The Mechanisms By Which RNA Helicase DDX5 Regulates REG3A MRNA Splicing To Promote Wound Healing In Diabetes

Reepithelialization is critical for wound healing,and impaired reepithelialization is one of the major reasons that contribute to impaired wound healing in diabetes.Our previous report has shown that REG3A can induce the proliferation of keratinocytes to promote wound reepithelializaton and regulate inflammatory responses in diabetes.However,the expression of REG3A is impaired in wounded skin of diabetes.Although we have shown that hyperglyceamia inhibits IL-33 to decrease the expression of REG3A,whether other mechanisms involved in regulating REG3A expression in skin wounds of diabetes remain unknown.It has been reported that the RNA helicase DDX5 plays an important role in RNA splicing,and regulates the proliferation and migration of keratinocytes.Whether DDX5 could regulate REG3A mRNA splicing to promote wound healing in diabetes remains unknown.In this study,we found that there were 3 variants of REG3A mRNA,and DDX5 promoted the expression of REG3A through increasing the variant 1 and variant 3 of REG3A mRNA by splicing the variant 2 of REG3A mRNA,and this process was also dependent on DDX17 and hnRNP H1.In addition,we observed that Reg?? mRNA variant 2 and the level of Ddx5 were decreased in wounded skin of diabetic mice.The deficiency of DDX5 in human keratinocytes led to decreased REG3A mRNA variant 1&3 but increased REG3A mRNA variant 2.All these data suggest that DDX5 regulate REG3A mRNA splicing to increase REG3A expression,thus promoting wound healing.To explore the mechanisms involved in the regulation of DDX5 in wounded skin of diabetes,we first check the mRNA expression of Ddx5 in wounded skin of normal and diabetic mice.We found that the mRNA expression of Ddx5 was not changed in wounded skin of diabetic mice compared to that of normal mice.This result indicated that the down-regulation of DDX5 in wounded skin of diabetes might be regulated by post-translational modification of DDX5.We then analyzed the SUMOylation of DDX5 and found that DDX5 was SUMOylated by both SUMO1 and SUM02/3,thus increas-ing the stability of DDX5.To identify which type of degradation of DDX5 would be prevented by the SUMOylation of DDX5,we evaluated whether DDX5 would be ubiquitinated.We confirmed that DDX5 was degraded via proteasome and the ubiquitination type of DDX5 was K27-linked ubiquitination,while the SUMOylation of DDX5 prevented this type of ubiquitination.Next we analyzed the SUMOylation-related genes in wounded skin,and found that multiple SUMOylation-related genes,especially the E3 ligase PIAS1,were up-regulated in wounded skin.Furthermore,we found that IL-17 was the key cytokine to induce PIAS1 expression in skin wounds,while hyperglycemia in diabetes inhibited IL-17 to induce PIAS1 expression in wounded skin of diabetes,thus decreasing DDX5 SUMOylation and its stability.In summary,our results demonstrate that DDX5 regulates REG3A mRNA splicing to increase REG3A expression,thus promoting wound healing.However,in diabetic skin wounds,the level of DDX5 is decreased due to impaired DDX5 SUMOylation that is regulated by PIAS1,thus leading to decreased REG3A expression and delayed wound healing in diabetes.Our findings reveal a new mechanism of the regulation of REG3A by DDX5,and provide a potential therapeutic target for treatment of diabetic wounds.