The Effect And Preliminary Analysis Of TsRNA Expression Profiles In Hyperbaric Oxygen Therapy For Diabetic Foot Ulcer

Objective: To investigate the alteration of tsRNA expression profile induced by HBO treatment in DFU and its functions.Methods: Clinical samples were divided into three groups: normal control group(Normal),diabetic foot ulcer group(DFU),hyperbaric oxygen group(HBO).Ulcer tissues were removed from the patients before and after they had undergone 14 consecutive days of HBO therapy sessions,whereas the control group was taken from the remaining normal skin of the above three patients at the time of surgical repair.Firstly,the tissue RNA was extracted by TRIzol method,and the RNA pretreatment and sequencing libraries were constructed.Then,we employed high-throughput sequencing technology to measure tsRNA expression levels and screen the differentially expressed genes in Normal,DFU,and HBO groups.Next,the differentially expressed tsRNAs were selected for real-time quantitative PCR(q PCR)validation.Furthermore,we used two common algorithms to predict tsRNA target genes,namely Targetscan and mi Randa,and validated by q PCR in tissue specimens.Finally,we carried out GO analysis and KEGG pathway analysis of the treatment-related tsRNA to predict potential functions.Results:1.High-throughput sequencing showed that a total of 422 tsRNAs were detected in the Normal,DFU and HBO specimens,and 220 tsRNAs were co-expressed in the three groups.According to the screening criteria of fold change >1.5,P<0.05,a total of 22 tsRNAs were significantly reversed after hyperbaric oxygen treatment.Compared with the normal group,there were five tsRNAs that were significantly downregulated in the DFU group but upregulated in the HBO group,whereas 17 tsRNAs were obviously upregulated in the DFU group but downregulated in the HBO group.2.Among these 22 significantly expressed tsRNAs,four significantly expressed tsRNAs were selected for q PCR validation,among which,t RF-Gly-CCC-006,t RF-Val-AAC-016 and t RF-Val-TAC-016 were statistically significant for inter-group comparison,which was consistent with the sequencing results.Therefore,these three tsRNAs were identified as tsRNAs associated with hyperbaric oxygen treatment for further analysis.3.Since tsRNAs can regulate the silencing of target genes in a mi RNA-like mediated manner,further q PCR validation was performed for WNT9 B,CREB3L2 and IGF1(corresponding to t RF-Gly-CCC-006,t RF-Val-AAC-016 and t RF-Val-TAC-016,respectively)among the predicted target genes,whose expression levels were opposite to the corresponding tsRNAs.4.GO analysis showed that differential tsRNAs were involved in molecular functions,such as cellular polysaccharide metabolism,UDP glycosyltransferase activity,molecular carrier activity and insulin-like growth factor 1 binding.the KEGG pathway enriched terms were highly relevant to the pathogenesis of DFU,such as Wnt,PI3K/Akt,MAPK and HIF-1.Conclusion: This study shows that tsRNA is differentially expressed in hyperbaric oxygen treatment of diabetic foot ulcers and may promote wound healing by regulating target genes in DFU-related signaling pathways,such as Wnt.