The Role And Mechanism Of Cold Shock Protein RBM3 In Diabetic Skin And Wound Healing

Objective: To explore the role and mechanism of cold shock protein RBM3 in diabetic skin and wound healing,so as to provide a new target for the treatment of refractory diabetic skin wounds,and broaden our understanding of the function of RBM3.Methods:1.In vivo experiment: Diabetic mouse model was induced by intraperitoneal injection of 50 mg/kg STZ for 5 consecutive days.Western blot and Immunohistochemistry were used to detect the protein expression of cold shock proteins RBM3 and CIRP in the skin tissues of mice after 1 month of successful diabetic model.RBM3 gene knockout mice and wild-type mice were established diabetes model,and then full-thickness skin defect wound model was established on the back of mice.Wound healing was observed and recorded at 0,3,5,7,9,11 days after injury,and the wound healing rate was calculated.2.In vitro experiment: Ha Cat cells were treated with different concentrations of D-glucose(5.5,10.5,25 and 40 m M)for 24 h,and the protein expression levels of RBM3 and CIRP were detected by Western blot.Ha Cat cells overexpressed with RBM3 were constructed by lentivirus infection,and then the Ha Cat cells were divided into four groups: empty-load + normal glucose,emptyload + high glucose,RBM3 overexpression + normal glucose,and RBM3 overexpression + high glucose(normal glucose: 5.5 m M;High glucose: 40 m M),CCK-8 assay,Scratch assay and Colony-forming assay were used to detect cell viability,migration ability,growth and colony formation ability,respectively.The protein expressions of AKT,m-TOR,p-m-TOR,ERK1/2 and p-ERK1/2were detected by Western blot.Finally,ERK1/2 inhibitor + high glucose were used to treat the control and RBM3 overexpression Ha Cat cells to observe the cell migration and growth.3.Transcriptome sequencing was performed on the control and RBM3 overexpressed Ha Cat cells,and gene expression was calculated to analyze the differentially expressed genes.The differentially expressed genes were enriched by GO and KEGG analysis.Results:1.Compared with the control group,the expression of RBM3 protein in the skin tissue of mice in the diabetic group was significantly increased(P<0.05),while the expression level of CIRP protein in both groups was low and the difference was not significant(P>0.05).However,in vitro studies,the expression of RBM3 protein in Ha Cat cells was decreased by high glucose treatment(P<0.05),while the expression of CIRP protein was not significantly changed(P>0.05).2.Overexpression of RBM3 can enhance the vitality of Ha Cat cells,promote cell growth and migration,and reverse the inhibition of high glucose on the proliferation and migration of Ha Cat cells to a certain extent.Overexpression of RBM3 can significantly increase the phosphorylation level of ERK1/2 in Ha Cat cells,especially under high glucose treatment(P<0.05),but has no significant effect on the AKT/m-TOR signaling pathway(P>0.05).After treatment with ERK1/2 selective inhibitor FR180204,RBM3’s ability to reverse the adverse effects of high glucose on Ha Cat cells disappeared.In addition,the deletion of RBM3 delayed the healing rate of dorsum skin wounds in mice,especially in the condition of high glucose.3.There were 224 differentially expressed genes in RBM3 overexpressed Ha Cat cells and control Ha Cat cells,among which 173 genes were significantly up-regulated and 51 genes were significantly down-regulated.These differentially expressed genes included genes related to CCL2 and AGE-RAGE signaling pathway,which play an important role in diabetes and its complications.GO and KEGG enrichment analysis showed that the overexpression of RBM3 in Ha Cat cells had so many effects.Conclusion:1.RBM3 can protect against the delayed healing of skin wounds caused by diabetes by promoting cell proliferation and migration.2.The mechanism of RBM3 promoting diabetic skin wound healing may be related to ERK1/2 signaling molecule.3.The overexpression of RBM3 in Ha Cat cells can cause changes in the transcription level of many genes,such as CCL2,AGE-RAGE and other genes that may be closely related to the healing of diabetic skin wounds.Therefore,RBM3 is a potential biomarker and therapeutic target of skin trauma in diabetes.