Screening Of Differentially Expressed CircRNAs,miRNAs In Diabetes Wounds And Study On The Mechanism Of Hsa＿circRNA＿000554 Participating In Wound Angiogenesis

Background:Diabetes wound is one of the common complications of diabetes.At present,the incidence rate of diabetes wound is increasing every year,and the number of patients with diabetes wound is about 1/4 of the total number of diabetes patients.Excessive local inflammatory response,impaired angiogenesis,abnormal accumulation of extracellular matrix and damaged epithelization of diabetes wound are significant pathophysiological changes in diabetes wound tissue,which lead to the failure recover of diabetes wound become to normal skin structure and function in a timely and orderly manner,diabetes wound often leads to intractable body infection or(and)amputation.Even if the medical personnel take strict blood glucose control and meticulous wound care,the prognosis of diabetes wounds is unfavourable.One of the key problems affecting the healing of diabetic wounds is the impaired vascular regeneration.CircRNAs(circular RNA)is one of endogenous non-coding RNA,which widely exists in eukaryotic cells.It is produced by RNA selective or reverse splicing and consists of exons and/or introns.It has a covalent closed ring structure,which is more stable than linear RNA.CircRNA has been shown to play a momentous role in the occurrence and development of various diseases.It can be used as a miRNA sponges to competitively bind miRNA,which will affect the expression of the target gene of miRNA.More and more evidences show that circRNAs and miRNAs are involved in the occurrence and development of tumors and endocrine and metabolic diseases.However,there are few literatures about the comprehensive analysis of circRNAs,miRNAs expression and competitive endogenous RNA(ce RNA)network in diabetes wound healing or vascular regeneration.This study firstly conducted a comprehensive analysis of circRNAs and miRNAs expression in diabetes wounds,screened miRNAs and circRNAs with abnormal expression and associated with vascularization for verification and ce RNA network map analysis.Then we applied vascular endothelial cell culture in vitro,Construction of overexpressed plasmid,transwell experiment,CCK-8,cell apoptosis experiment,scratch experiment,tubule formation experiment,double luciferase reporter gene experiment and other methods to systematically observate the effects of hsa＿circRNA＿000554 on the biological functions of downstream miRNAs and preliminarily explore the possible molecular mechanism of hsa＿circRNA＿000554 in angiogenesis of diabetes wounds.Part I Analysis of circRNAs expression profile in diabetes wounds Object:The differential expression of circRNAs in diabetic wound and normal skin tissue were screened and bioinformatics analysis was conducted to provide a basis for searching for the possible molecular mechanism of vascular regeneration in diabetic wound.Methods:The diabetes wound tissues and normal skin tissues of 16 patients were collected,and the expression profiles of circRNA in diabetes wound tissues and normal skin tissues were obtained by circRNA chip.CircRNAs with up-regulated or down-regulated fold changes greater than 2 were selected.GO and KEGG analysis were performed on the differentially expressed circRNAs to find out the signal pathway that circRNAs are significantly enriched in diabetes wound healing.q RT-PCR was used to detect the expression changes of circRNAs with significant expression differences in diabetic wounds.Results:The results of circRNA microarray analysis showed that there were 356 circRNAs with multiple expression differences greater than 2.The 157 circRNAs were up-regulated and 199 circRNAs were down-regulated.The GO and KEGG analysis of differentially expressed circRNAs showed showed that they were mainly related to MAPK,AGE-RAGE,acute myeloid leukemia,RNA transport,B cell receptor signal pathway,human cytomegalovirus infection,and vascular smooth muscle contraction-related pathways.The host genes of these differentially expressed circRNAs are mainly related to cancer,infectious diseases,endocrine and metabolism related diseases,neurodegenerative diseases,etc.in human diseases.They are highly related to the endocrine system,immune system,nervous system,development in the body system.In the cell process,they are significantly related to cell proliferation and apoptosis,cell community eukaryotes,and cell transport and catabolism.It is related to signal transduction and interaction of signal molecules in environmental information processing,transformation in genetic information processing,and distribution in metabolism.In addition,KEGG analysis showed that the dysregulated circRNA was related to the regulation of vascular smooth muscle contraction-related pathways and actin cytoskeleton.The up-regulated circRNA-001588 and circRNA-104330 and down-regulated circRNA-000554,circRNA-101280,circRNA-100451 and circRNA-103107 were detected by q RT-PCR.Conclusions:There are differences in circRNAs expression between diabetes wounds and normal skin tissues.The disordered circRNAs are related to the contraction related pathways of vascular smooth muscle and the regulation of actin cytoskeleton.Based on these data,the biological function of these circRNAs may contribute to the study of angiogenesis in chronic wounds of diabetes.Part II Analysis of miRNAs expression profile in diabetes wounds Object:The differential expression of miRNAs in diabetic wound and normal skin tissue were screened and bioinformatics analysis was conducted to provide a basis for searching for the possible molecular mechanism of vascular regeneration in diabetic wound.Methods:The wound tissues and normal skin tissues of 16 patients with diabetes were collected.RNA high-throughout sequencing was used to obtain the miRNA expression profile in diabetes wound tissues and normal skin tissues.The miRNAs whose expression was up-regulated or down-regulated by more than 2 times were screened out.The differentially expressed miRNAs were analyzed by GO and KEGG using the bioinformatics software Target Scan,mi-Randa and miRbase.The expression changes of 7 miRNAs associated with vascularization in diabetic wounds were detected by q RT-PCR.The 11 circRNAs and 48 miRNAs that were screened and related to diabetic wound healing were analyzed by ce RNA network diagram.Results:The results of RNA high-throughput sequencing showed that there were 42 miRNAs with multiple expression differences greater than 2,including 2 up-regulated miRNAs and 40 down-regulated miRNAs.The hsa-miRNA-223-3p,hsa-miRNA-114-3p,hsa-miR-144-3p,hsa-miRNA-205-5p,hsa-miRNA-200c-3p,hsa-miRNA-141-3p and hsa-miRNA-514a-39 were detected by q RT-PCR.The GO and KEGG analysis results of 42 miRNAs showed that they were mainly related to intercellular metabolic regulation,metabolic regulation,biological positive regulation,morphological formation of cell anatomical structure,nervous system development,positive metabolic regulation,intercellular signal transduction regulation,DNA-binding transcription factor activity,intracellular metal ion binding,cation binding,transcription factor activity regulation,RNA polymerase II transcription factor activity,intracellular transfer enzyme activity Protein binding and other biological functions are related,and most of them are involved in NOD-like receptor signaling pathway,Fanconi anemia pathway,TNF signaling pathway,Cysteine and methionine metabolism,Autophagy,ECM-receptor interaction,Photoshatidylinositol signaling system,NF-κB signaling pathway,signaling pathways regulating pluripotency of stem cells,Cell adhesion molecules(CAMs)TGF-β signaling and other pathways.The ce RNA showed that hsa＿circRNA＿000554 could bind miR-144-3p specifically,and hsa＿circRNA＿100141 and hsa＿circRNA＿103107 could competitively bind miR-203a-3p.hsa＿circRNA＿001588 and hsa＿circRNA＿104330can jointly compete with miR-514a-3p binding sites.hsa＿circRNA＿100926,hsa＿circRNA＿100927,and hsa＿circRNA＿101280 competitively bind to miR-429.Conclusions:There are differences in the expression of miRNAs in diabetes wound and normal skin tissue,they are mostly involved in the regulation of NOD like receptor signaling pathway,Fanconi anemia pathway,TNF signaling pathway,Autophagy,ECM receiver interaction,NF kappa B signaling pathway,signaling pathways regulation,TGF beta signaling and other pathways.Abnormally expressed circRNAs and miRNAs have a network regulatory relationship with each other.Part Ⅲ Mechanism of hsa＿circRNA＿000554 regulating vascular regeneration in diabetic wound Object:To explore the mechanism of hsa＿circRNA＿000554 regulating vascular endothelial cell regeneration through miR-144-3p/VEGF-A axis.Methods:Double luciferase reporter gene experiment was performed to verify the specific binding of hsa＿circRNA＿000554 to miR-144-3p and miR-144-3p to VEGF-A.The constructed overexpressed hsa＿circRNA＿000554 plasmid and blank control plasmid were transfected into vascular endothelial cells,and the differential expression of hsa＿circRNA＿000554,miR-144-3p and VEGF-A in the two groups were detected by q RT-PCR.The expression level of VEGF-A in vascular endothelial cells transfected with miR-144-3p mimic was detected by q RT-PCR.q RT-PCR was used to detect the expression of VEGF-A in vascular endothelial cells of blank control group,overexpressed hsa＿circRNA＿000554 group,and co-transfected group of hsa＿circRNA＿000554 +miR-144-3p mimic(Recovery experiment).The effects of hsa＿circRNA＿000554 on the proliferation,migration,apoptosis and in vitro tubular formation of vascular endothelial cells were defined by CCK-8,Transwell migration assay,apoptosis assay and tubule formation assay.Results:Double luciferase reporter gene assay showed that hsa＿circRNA＿000554 could specifically bind to miR-144-3p,and miR-144-3p could specifically bind to VEGF-A.Compared with blank control group,the expressions of hsa＿circRNA＿000554 and VEGF-A in hsa＿circRNA＿000554 overexpression group were significantly increased,while the expression of miR-144-3p was significantly decreased(p<0.01).Compared with miR-144-3p mimic NC group,the expression of VEGF-A in miR-144-3p mimic group was significantly decreased(p<0.05).Compared with blank control group,the overexpression of hsa＿circRNA＿000554 significantly increased the expression of VEGF-A(p<0.01),and the effect of hsa＿circRNA＿000554 overexpression on VEGF-A was adjusted by miRNA-144-3p mimic(p<0.001).Compared with the control group,overexpression of hsa＿circRNA＿000554 promoted the proliferation,migration and in vitro angiogenesis of vascular endothelial cells,and inhibited cell apoptosis(p<0.05).Conclusions:hsa＿circRNA＿000554 specifically binds miR-144-3p,which can specifically bind VEGF-A.hsa＿circRNA＿000554 can change the expression of VEGF-A in vascular endothelial cells by regulating miR-144-3p,thus affecting the proliferation,migration and in vitro angiogenesis of vascular endothelial cells.hsa＿circRNA＿000554/miR-144-3p/VEGF-A axis may be a regulatory mechanism affecting vascular regeneration in diabetic wound.The total Conclusion:1.Compared with normal skin tissue,circRNA and miRNA differentially expressed in diabetic wounds are more involved in the regulation of AGE-RAGE,NF-κB,Wnt/β-Catenin and other signaling pathways,which are closely related to the angiogenesis of diabetic wound.2.Overexpression of hsa＿circRNA＿000554 promoted the proliferation,migration and angiogenesis of vascular endothelial cells cultured in vitro,and inhibited the apoptosis of vascular endothelial cells.hsa＿circRNA＿000554 can change the intracellular VEGF-A content by regulating miR-144-3p,and affect the process of vascular regeneration in diabetic wound.