Studies Of The Impact And Mechanisms Of MiR-103a-3p Targeting RCAN1 On The Function Of Human Dermal Fibroblasts Under High-Glucose Stimulation

Research background Diabetic foot ulcers(DFUs)are one of the most severe complications of diabetes,leading to significant disability and mortality among diabetic patients.DFU patients experience impaired wound healing due to abnormal inflammatory responses in wound tissues,alterations in the migration and proliferation of fibroblasts and keratinocytes,and compromised blood vessel formation.Therefore,the inquiry for mechanisms that promote DFU wound healing is of paramount importance for improving DFU prognosis.Micro RNAs(mi RNAs)are a class of endogenous,single-stranded non-coding RNAs.They can modulate multiple gene targets and signaling pathways that influence the healing of DFUs,making them valuable players in the process associated with DFU wound healing.Mi R-103a-3p,as a member of the mi RNA family,has been found to be closely linked to diabetes.Through screening mi R-103a-3p target genes using Target Scan and star Base databases,and conducting GO and KEGG enrichment analysis of the mi R-103a-3p target gene set,we discover that RCAN1 is a target gene of mi R-103a-3p.RCAN1 can be enriched in the PI3K/Akt signaling pathway,through which it can regulate cell survival and apoptosis.However,to date,there have been no reports on the role and mechanisms of mi R-103a-3p in the healing of diabetic foot ulcers.This study aims to investigate the potential role and mechanisms of mi R-103a-3p in the wound healing process of DFUs.The research consists of two parts:(i)The expression and clinical significance of mi R-103a-3p and RCAN1 in diabetic foot ulcer tissues.(ii)The impact of mi R-103a-3p targeting RCAN1 on the function of human dermal fibroblasts under high glucose stimulation,along with a study of the underlying mechanisms.Expressions and Clinical Significance of Mi R-103a-3p and RCAN1 in Diabetic Foot Ulcer TissuesPurpose(i)To determine the expression levels of mi R-103a-3p and RCAN1 in diabetic foot ulcer tissues and further analyze their relationships with clinical parameters of diabetic foot ulcers.(ii)To investigate the clinical significance of mi R-103a-3p and RCAN1 in the wound healing process of diabetic foot ulcers.Method1.Selecting 60 diabetic foot ulcer patients as the experimental group and 30non-diabetic patients with chronic foot ulcers as the control group,we collect tissue samples from the ulcer edges and peripheral blood of these patients;2.Clinical parameters are assessed in the study subjects as follows: 1)Venous blood collection: fasting plasma glucose(FPG),glycosylated hemoglobin A1c(Hb A1c),white blood cell count(WBC),C-reactive protein(CRP);2)Other examinations: skin ulcer area and ankle brachial index(ABI);3)General patient information recording: For the DFU group(age,gender,duration of illness,wagner classification,and ulcer healing rate)and for the control group(age,gender and duration of illness)3.The expressions of mi R-103a-3p and RCAN1 genes in the ulcer edge tissues of the study subjects are determined using real-time quantitative PCR(RT-q PCR).The expression of RCAN1 protein in the ulcer edge tissues is assessed using Western blot analysis(WB).The protein expressions and localization of RCAN1 and Bax in the ulcer edge tissues are detected using immunohistochemistry(IHC).The protein expressions of tumor necrosis factor-α(TNF-α)and interleukin-6(IL-6)in the peripheral blood of the study subjects are measured using enzyme-linked immunosorbent assay(ELISA).4.The correlations between the expression of mi R-103a-3p and RCAN1,Bax,IL-6,TNF-α,and various clinical parameters in the DFU group and the control group are analyzed.Results1.Comparison of clinical parameters between DFU and control groups: There are no statistically significant differences in gender distribution,age,disease duration,and ulcer area between the DFU group and the control group(P >0.05).The DFU group has significantly higher levels of FPG,Hb A1 c,CRP,and WBC compared to the control group,while the ABI level is lower,and these differences are statistically significant(P<0.05).2.Comparison of clinical characteristics between the high and low expression groups of mi R-103a-3p in the DFU group’s foot ulcer edge tissues: In the DFU group,the high expression group of mi R-103a-3p exhibits a lower ulcer healing rate at 4weeks compared to the low expression group,and this difference is statistically significant(P=0.006).This suggests a close association between the expression level of mi R-103a-3p in foot ulcer edge tissues and the ulcer healing rate at 4 weeks.3.Expression of mi R-103a-3p in the wound edge tissue of DFU group and control group: The expression level of mi R-103a-3p in the wound edge tissue of the DFU group is higher than that in the wound edge tissue of the control group(2.928±0.912 vs.1.025±0.712,P <0.05).4.Expression of RCAN1 mRNA and protein in the wound edge tissue of DFU group and control group: The expression levels of RCAN1 m RNA and protein in the wound edge tissue of the DFU group are both lower than those in the wound edge tissue of the control group(m RNA: 0.516±0.302 vs 1.012±0.372,P<0.05;protein:0.391±0.153 vs 0.645±0.162,P<0.05).5.Expression and localization of RCAN1 and Bax proteins in the wound edge tissue of DFU group and control group: Under electron microscopy,RCAN1 and Bax proteins are stained brown.Their positive expression is mainly concentrated in the cytoplasm of fibroblasts,vascular endothelial cells,and macrophages.The expression of RCAN1 protein in the wound edge tissue of the DFU group is slightly lower than that in the wound edge tissue of the control group(0.535±0.071 vs 0.711±0.089,P <0.05);however,the expression of Bax protein in the wound edge tissue of the DFU group is higher than that in the wound edge tissue of the control group(2.945±0.102 vs1.174±0.099,P <0.05).6.Expression of TNF-α and IL-6 proteins in the serum of DFU group and control group patients: The protein expression level of TNF-α in the serum of DFU group patients is higher than that in the control group(31.275±10.246 pg/ml vs 10.543±4.857pg/ml,P <0.05).Similarly,the protein expression level of IL-6 in the serum of DFU group patients is higher compared to the control group(22.354±4.868 pg/ml vs7.876±2.915 pg/ml,P < 0.05).7.Correlation between mi R-103a-3p expression levels in the wound edge tissue of the DFU group and control group and various parameter indicators: In the wound edge tissue of the DFU group,the expression level of mi R-103a-3p is negatively correlated with RCAN1 m RNA and ABI levels(r =-0.412 and-0.108,respectively,both P <0.05).It is positively correlated with peripheral blood FPG,Hb A1 c,CRP,TNF-α,IL-6 proteins,and Bax protein levels(r = 0.356,0.329,0.572,0.698,0.473,and 0.579,respectively,all P <0.05).In the wound edge tissue of the control group,mi R-103a-3p expression level is negatively correlated with RCAN1 m RNA level(r =-0.391,P < 0.05)and positively correlated with TNF-α in peripheral blood level and Bax protein level in wound edge tissue(r= 0.399 and 0.421,respectively,both P <0.05).Conclusions1.The expression level of mi R-103a-3p in the wound edge tissue of the DFU group is higher compared to the control group.Furthermore,there is a negative correlation between mi R-103a-3p expression and RCAN1 expression levels,suggesting a potential association between mi R-103a-3p and RCAN1.2.The expression level of mi R-103a-3p in the wound edge tissue of the DFU group shows a positive correlation with blood glucose,pro-inflammatory cytokines,apoptosis-related proteins levels.This suggests that mi R-103a-3p may potentially be involved in the wound healing process of DFU by influencing inflammatory responses,and the apoptosis pathway.Studies of the Impact and Mechanisms of Mi R-103a-3p Targeting RCAN1 on the Human Dermal Fibroblasts Function Under High-Glucose StimulationPurpose(i)To confirm mi R-103a-3p can target and regulate RCAN1.(ii)To confirm that mi R-103a-3p targeting RCAN1 affects apoptosis,migration,and inflammatory response in human dermal fibroblasts under high glucose stimulation.(iii)To investigate the molecular mechanism by which mi R-103a-3p targets RCAN1 to regulate the functions of human dermal fibroblasts.Method1.Culturing human dermal fibroblasts,performing cell passaging,cryopreservation,and revival.2.Purchase of mi R-103a-3p mimics(mimics-mi R-103a-3p)and its negative control(mimics-NC),mi R-103a-3p inhibitor(inhibitor-mi R-103a-3p)and its negative control(inhibitor-NC),si-RCAN1 and its negative control(si-NC),RCAN1 overexpression vector(OE-RCAN1),and its negative control empty vector(OE-NC).3.Three types of si RNA and the negative control group are designed and transfected into NHDFs.Additionally,a normal cell group is set as an another control group.The silencing effect of these three si RNAs is validated by RT-q PCR,comparing the RCAN1 m RNA expression levels in cells treated with si RNAs to those in normal cell group and the negative control group.4.RCAN1 3’-UTR-WT vectors or RCAN1 3’-UTR-Mut vectors(purchased externally)is co-transfected with mimics-mi R-103a-3p or mimics-NC into NHDFs.After transfection,a dual-luciferase reporter gene detection system is used to assess the relationship between RCAN1 and mi R-103a-3p.5.NHDFs are cultured in high-glucose medium for different durations(12h,24 h,48h,72h).Flow cytometry is applied to analyze the apoptosis changes in NHDFs under high-glucose stimulation,selecting the optimal time for high-glucose intervention in NHDFs for subsequent experiments.Cell supernatants are collected,and ELISA is used to measure the levels of the pro-inflammatory cytokines TNF-α and IL-6.6.Various intervention experiments in high-glucose cultured NHDFs:1)Related experiments involving overexpression or inhibition of mi R-103a-3p(experimental groups: NC group,HG group,HG+mimics-mi R-103 group,HG+mimics-NC group,HG+inhibitor-mi R-103 group,HG+inhibitor-NC group).2)Related experiments involving overexpression or silencing of RCAN1(experimental groups: NC group,HG group,HG+OE-RCAN1 group,HG+OE-NC group,HG+si-RCAN1 group,HG+si-NC group).3)Related experiments involving inhibition of mi R-103a-3p and silencing of RCAN1(experimental groups: NC group,HG group,HG+si-NC+inhibitor-NC group,HG+si-RCAN1+inhibitor-NC group,HG+si-NC+inhibitor-mi R-103 group,HG+si-RCAN1+inhibitor-mi R-103 group).4)Related experiments involving overexpression of RCAN1 and PI3 K inhibitor intervention(experimental groups: HG+OE-RCAN1 group,HG+OE-NC group,HG+OE-NC+PI3K inhibitor group,HG+OE-RCAN1+PI3K inhibitor group).Flow cytometry is used to assess apoptosis changes in NHDFs under high glucose stimulation.Cell scratch assays are employed to investigate migration changes in NHDFs under high glucose stimulation.RT-q PCR and Western blotting are utilized to examine the expression changes of PI3K-Akt-Bad-related m RNA and proteins in NHDFs under high glucose stimulation.ELISA is applied to measure the expression changes of TNF-α and IL-6 proteins in NHDFs under high glucose stimulation.Results1.Dual-luciferase reporter gene experiment verification: RCAN1 has binding sites with mi R-103a-3p,confirming it as the target gene of mi R-103a-3p.2.Si RNA screening experiment: si-RNA3 exhibits the highest silencing efficiency among the tested si RNAs,and it is chosen for further experiments.3.Effects of high glucose intervention on NHDFs apoptosis and pro-inflammatory cytokine expression over time: With the passage of time under high glucose intervention,the apoptosis and necrosis rates of NHDFs increase,showing time dependency.The protein expression levels of TNF-α and IL-6 gradually increase in NHDFs before 48 hours of high glucose intervention,follow by a gradual decrease after48 hours.4.Effects of mi R-103a-3p on NHDFs function,PI3K-Akt-Bad signaling pathway,and pro-inflammatory cytokine expression under high glucose stimulation:Compared to the NC group,the HG group shows that the apoptosis rate of NHDFs,Bad,Bax,TNF-α and IL-6 expression levels increase,while migration rate of NHDFs,PI3 K,p-Akt,p-Bad,and Bcl-2 expression levels decrease under high glucose stimulation.Compared to the HG+mimics-NC group,the HG+mimics-mi R-103 group shows that the apoptosis rate of NHDFs,Bad,Bax,TNF-α and IL-6 expression levels increase,while migration rate of NHDFs,PI3 K,p-Akt,p-Bad,and Bcl-2 expression levels decrease under high glucose stimulation.Compared to the HG+inhibitor-NC group,the HG+inhibitor-mi R-103 group shows that the apoptosis rate of NHDFs,Bad,Bax,TNF-α and IL-6 expression levels decrease,while migration rate of NHDFs,PI3 K,p-Akt,p-Bad,and Bcl-2 expression levels increase under high glucose stimulation.5.Effects of RCAN1 on NHDFs function,PI3K-Akt-Bad signaling pathway,and pro-inflammatory cytokine expression under high glucose stimulation: Compared to the HG+OE-NC group,the HG+OE-RCAN1 group shows that the apoptosis rate of NHDFs,Bad,Bax,TNF-α and IL-6 expression levels decrease,while migration rate of NHDFs,PI3 K,p-Akt,p-Bad,and Bcl-2 expression levels increase under high glucose stimulation.Compared to the HG+si-NC group,the HG+si-RCAN1 group shows that the apoptosis rate of NHDFs,Bad,Bax,TNF-α and IL-6 expression levels increase,while migration rate of NHDFs,PI3 K,p-Akt,p-Bad,and Bcl-2 expression levels decrease under high glucose stimulation.6.Effects of inhibiting mi R-103a-3p and silencing RCAN1 on NHDFs function,PI3K-Akt-Bad signaling pathway,and pro-inflammatory cytokine expression under high glucose stimulation: Compared to the HG+si-RCAN1+inhibitor-NC group,the HG+ si-RCAN1+inhibitor-mi R-103 group shows that the apoptosis rate of NHDFs,Bad,Bax,TNF-α and IL-6 expression levels decrease,while migration rate of NHDFs,PI3 K,p-Akt,p-Bad,and Bcl-2 expression levels increase under high glucose stimulation.Compared to the HG+si-NC+inhibitor-mi R-103 group,the HG+si-RCAN1+inhibitor-mi R-103 group shows that the apoptosis rate of NHDFs,Bad,Bax,TNF-αand IL-6 expression levels increase,while migration rate of NHDFs,PI3 K,p-Akt,p-Bad,and Bcl-2 expression levels decrease under high glucose stimulation.7.Effects of overexpressing RCAN1 and PI3 K inhibitor intervention on NHDFs function,PI3K-Akt-Bad signaling pathway,and pro-inflammatory cytokine expression under high glucose stimulation: Compared to the HG+OE-NC group,the HG+OE-NC+PI3K inhibitor group shows that the apoptosis rate of NHDFs,Bad,Bax,TNF-α and IL-6 expression levels increase,while migration rate of NHDFs,PI3 K,p-Akt,p-Bad,and Bcl-2 expression levels decrease under high glucose stimulation.Compared to the HG+OE-RCAN1 group,the HG+OE-RCAN1+PI3K inhibitor group shows that the apoptosis rate of NHDFs,Bad,Bax,TNF-α and IL-6 expression levels increase,while migration rate of NHDFs,PI3 K,p-Akt,p-Bad,and Bcl-2 expression levels decrease under high glucose stimulation.Compared to the HG+OE-NC+PI3K inhibitor group,the HG+OE-RCAN1+PI3K inhibitor group shows that the apoptosis rate of NHDFs,Bad,Bax,TNF-α and IL-6 expression levels decrease,while migration rate of NHDFs,PI3 K,p-Akt,p-Bad,and Bcl-2 expression levels increase under high glucose stimulation.Conclusions1.RCAN1 is a target gene of mi R-103a-3p,and mi R-103a-3p negatively regulates the expression of RCAN1.2.High glucose promotes apoptosis of human dermal fibroblasts,inhibits their migration,and stimulates the expression of pro-inflammatory cytokines.3.Inhibiting the expression of mi R-103a-3p and overexpressing RCAN1 both attenuates the damage to human dermal fibroblasts caused by high glucose.4.Interfering with RCAN1 expression reduces the protective effect of inhibiting mi R-103a-3p expression on human dermal fibroblasts in a high-glucose environment.5.Mi R-103a-3p targeting RCAN1 mediates the role of the PI3K/Akt signaling pathway in regulating apoptosis,migration,and the expression of pro-inflammatory cytokines in human dermal fibroblasts.