| Background and objective: Pemphigus is a life-threatening autoimmune bullous disease.The typical skin lesions are flaccid blisters on normal skin or mucous membrane and postbullous erosion.Histopathology is characterized by acantholysis of the upper basal layer or granular layer in the epidermis.Pemphigus vulgaris(PV)and pemphigus foliaceus(PF)are common subtypes of pemphigus.The recognized pathogenesis is due to the presence of pathogenic autoantibodies against keratinocyte surface structure,desmoglein(Dsg)1 and/or Dsg3,which cause antigen-antibody reaction,resulting in structural changes of desmosomes,loss of intercellular adhesion,acantholysis and blister formation.MicroRNAs(miRNAs) are non-coding small RNAs.As the gene regulators,miRNAs bind to the 3’UTR region of the target genes,degrade the target m RNAs or inhibit the translation of the target genes,and play an important role in many biological functions such as cell proliferation,migration,differentiation,apoptosis and so on.One miRNA can regulate multiple target genes at the same time,and one gene can also be regulated by multiple miRNAs.Mi RNAs can be expressed in tissues,but also enter the humoral circulation,and stably exist in blood and other body fluids as circulating miRNAs.Many reports have shown that miRNAs play important roles in the pathogenesis,development and response to treatment in various diseases.Circulating miRNAs have been proved to be biomarkers of many diseases,which can be used in disease diagnosis,staging,differential classification,reflecting the effect of treatment and evaluating prognosis.In recent years,more evidences have shown that the dysregulated of miRNA expression can cause immune dysfunction,break immune tolerance and lead to autoimmunity.Micro RNAs have been proved to play an important role in the development,maintenance of immune homeostasis and normal function of the immune system.Circulating miRNAs can be used as biomarkers of autoimmune diseases,such as systemic lupus erythematosus,rheumatoid arthritis,systemic sclerosis and so on.At present,there are few reports about pemphigus-related miRNA.Only a few studies have reported the expression of miRNA in peripheral blood monocytes of patients with pemphigus.However,whether the expression of miRNA in serum has changed during the pathogenesis of pemphigus it is not clear.The purpose of this study was to detect the serum miRNA expression profile of patients with pemphigus and find the differentially expressed miRNA.Materials and methods:1.Subjects: the serum samples of this study were collected from patients in the first affiliated Hospital of China Medical University.A total of 53 pemphigus patients with active pemphigus hospitalized in the department of dermatology from December 2016 to December 2019.The diagnosis was made by typical clinical manifestations,histopathological examination,direct immunofluorescence,indirect immunofluorescence and positive anti-Dsg1 and/or anti-Dsg3 antibodies.A total of 27 age and gender matched normal subjects were selected as healthy controls.2.The serum microRNA expression profiles of 8 PV patients,8 PF patients and 8 healthy controls were detected by Taqman Micro RNA Array.3.The serum samples of 19 PV patients,PF 18 patients and 19 healthy controls were selected to verify the difference of micro RNA by q PCR.4.The serum levels of anti-Dsg1 antibody and anti-Dsg3 antibody were detected by ELISA in 19 PV patients 18 PF patients and 19 healthy controls.5.The target genes were predicted by bioinformatics method.6.The target genes were verified by Dal-Luciferase reporter assay.7.The m RNA levels of miRNA mimic or NC transfected Ha Ca T cells were detected by RT-q PCR assay.8.The protein levels of miRNA mimic or NC transfected Ha Ca T cells were detected by western blot assay.9.Statistical analysis: All statistical analyses were performed using Graph Pad Prim and SPSS softwares.Data are expressed as median(IQR).Mann-Whitney U test was applied for two groups comparion.Wilcoxon signed-rank test was applied for paired data.Two tailed P < 0.05 was considered as significant difference.Results:1.Totally 20 upregulated and 27 downregulated miRNAs were observed in the serum of the 8 PV patients in comparison with the 8 healthy controls.2.Compared with the healthy controls,the serum expressions of let-7e-5p,miR-26b-5p and miR-374a-5p in PV patients were significantly up-regulated by q PCR.3.The diagnostic efficiency of serum let-7e-5p,miR-26b-5p and miR-374a-5p in patients with PV was evaluated by ROC curve,and the AUC values were 0.765,0.726 and 0.723,respectively.It was considered that let-7e-5p,miR-26b-5p and miR-374a-5p had certain diagnostic value for PV.4.The relative expression of serum let-7e-5p in patients with PV was significantly increased in the disease control stage.5.A total of 43 differential miRNA were screened in PF group,of which 25 were up-regulated and 18 down-regulated.6.q PCR analysis showed that the relative expressions of miR-26b-5p and miR-320 b in serum of patients with PF were significantly down-regulated.7.The diagnostic efficiency of serum miR-26b-5p and miR-320 b in PF was evaluated by ROC curve,and the AUC values were 0.702 and 0.737,respectively.Serum miR-26b-5p and miR-320 b had certain diagnostic value for PF.8.No trend was found in the relative expression of serum miR-26b-5p and miR-320 b in patients with PF during the active stage and the disease control stage.9.Hsa-let-7e-5p significantly down-regulated the luciferase expression of h-DSG3-3UTR-WT.10.The expression of DSG3 protein decreased in Ha Ca T cells transfected with let-7e-5p mimic.Conclusion: 1.The levels of let-7e-5p,miR-26b-5p,and miR-374a-5p in serum of patients with PV was significantly down-regulated.2.The level of serum let-7e-5p in patients with PV was significantly up-regulated after effective treatment.3.Serum let-7e-5p may be a biomarker to reflect the activity and treatment response of PV patients.4.The levels of miR-26b-5p and miR-320 b in serum of patients with PF were significantly down-regulated.5.Let-7e-5p targets to DSG3 3’UTR,interferes with the translation of DSG3 and regulates the protein expression of DSG3. |
PDF Full Text Request