| Research BackgroundDermatology covers a large amount of diseases and there are around 2000-3000 kinds of disease been defined for now.However,life-threatening ones accounts for only 2%of these diseases,and pemphigus is one of them.Pemphigus is an autoimmune disease whose mechanism is not fully understood.The current view is that it is an autoantibody related disease.The main onset age of pemphigus is 50-60 years old,and the incidences between men and women are equal.The main symptoms of pemphigus are skin and mucosal blisters and erosions[1].Due to the damage of skin barrier,the risk of infection is greatly increased in these patient.Moreover,the erosions of the skin and mucous membranes make the patients suffer from itching and paining.So that they can not eat and sleep comfortably,which may cause a sharp decrease of life quality as a result.Thus,receiving early diagnosis and appropriated treatment in time are very important for pemphigus patients.Bullous related diseases are classified into many species,including pemphigus,paraneoplastic pemphigus(PNP),familial benign chronic pemphigus(hailey hailey disease,HHD),bullous pemphigoid(BP),cicatricial pemphigoid,dermatitis herpetiformis,linear IGA bullous dermatosis,epidermolysis bullosa acquisita(EBA),epidermolysis bullosa dystrophica(EBD)and so on.Though they have similar clinical presentation,they are different types of diseases and have different treatment methods and priorities.Most of time,we have to distinguish pemphigus with BP,as the latter has higher incidence and shares the same onset age with pemphigus.Currently,the main diagnostic criteria for pemphigus includes histopathology and immunofluorescence.As a matter of fact,the traditional diagnostic methods are cumbersome and time-consuming,which is not conducive to clinical diagnosis and treatment.Therefore,it is urgent to discover a new accurate and rapid biological diagnosis indicators to guide the early diagnosis.MicroRNAs(miRNAs)are endogenous small RNAs that play an important role in gene transcription and expression,which are involved in many biological functions such as cell proliferation,differentiation,metastasis and apoptosis.A number of autoimmune-related diseases such as systemic lupus erythematosus(SLE),multiple sclerosis(MS),rheumatoid arthritis(RA)and psoriasis were found to be significantly correlated with abnormal expressions of some miRNAs[2].MiR-338-3p has recently been found to play a regulatory role in the body as a tumor suppressor.The expression level of miR-338-3p in tumor tissue was significantly decreased.Consistently,artificially up-regulated the expression of miR-338-3p could inhibit the proliferation,migration and promote the apoptosis of tumor cells[3,4].Nevertheless,the regulatory function of miR-338-3p in non-tumor cells or tissues remains unknown.We found that the expression level of miR-338-3p was significantly up-regulated in PBMCs of pemphigus patient through our previous gene chip analyzation,which implys that there may be some regulatory functions and potential biological diagnostic value of miR-338-3p.In this study,peripheral blood mononuclear cells(PBMCs)from patients with active pemphigus,inactive pemphigus and active BP were isolated to detect the expression of miR-338-3p through Real-time quantitative Polymerase Chain Reaction(RT-qPCR).Patients with active pemphigus were followed up for at least 2 weeks to monitor changes of miR-338-3p expression in PBMCs.Finally,we up-regulated the expression of miR-338-3p of PBMC isolated from health control and then screened the potential target genes and investigated their function.Purpose1.To confirm whether the expression of miR-33 8-3p in patients with active pemphigus is elevated.Is there any differences of the expression level of miR-338-3p between patients with active pemphigus,inactive pemphigus,active bullous pemphigoid and health controls?2.To detect changes of the expression of miR-338-3p during the treatment of pemphigus.To investigate whether the expression of miR-338-3p can indicate treatment effectiveness.To further assess the associations between expression of miR-338-3p and disease severity of pemphigus.3.To screen for possible target genes of miR-338-3p and perform functional studies.Method1.Blood samples of patients with active pemphigus,inactive pemphigus and active BP were collected from outpatients and wards at our department.Blood samples of healthy controls were collected at the Health Management Center of our hospital.PBMCs were isolated,the expression of miR-338-3p was detected by RT-qPCR and the expression level of miR-338-3p in different groups were compared.Pemphigus Disease Area Index(PDAI)and Autoimmune Bullous Skin Disorder Intensity Score(ABSIS)of patients with active pemphigus were collected.The patients were divided into different group according to severity,gender and whteher relapse,and the associations with the assessed scores and the level of miR-338-3p among different groups were analyzed.2.Patients with active pemphigus who accepted treatment were followed up for at least 2 weeks.Basic clinical examination information was collected,both PDAI and ABSIS were evaluated regularly.The serum level of pemphigus antibodies and the PBMC miR-338-3p expression level before and after treatment were tested and compared.After adjusting for baseline characteristics and other clinical parameters,the associations between expression level of miR-338-3p and disease severity of pemphigus,which is reflected by PDAI scores and ABSIS scores,was further evaluated.3.PBMCs from health controls were isolated and transfected so as to up-regulate the expression of miR-338-3p.Target genes were screened through PCR and confirmed through western blot and luciferase reporter gene.Statistical analysisWe performed all the statistical analyses using SPSS20.0 software.Continuous variables were expressed as mean±standard deviation(x±SD).Categorical variables were expressed as frequencies and percentages(n,%).Statistical methods used in this study included independent samples t-test,paired samples t-test,one-way ANOVA,Receiver Operating Characteristic(ROC)Analysis,Chi-square test,Fisher exact test and Generalized Estimating Equation(GEE).Relative expression of miR-338-3p was calculated using 2-△△CT equation(△ CT value=CT value of target gene-CT value of reference gene;△△ CT value=△ CT value of experimental group-△CT value of control group).A value of P<0.05 was considered statistically significant.Result1.The expression level of miR-338-3p in PBMC of patients with active pemphigus was significantly higher than health control group,patients with inactive pemphigus group and patients with active bullous pemphigoid group.The expression level of miR-338-3p in patients with active pemphigus has no differences among subgroups defined by gender,disease severity,disease condition.2.The expression of miR-338-3p decreased with the improvement of the disease in patients with active pemphigus after treatment.The miR-338-3p expression level was compared before treatment,2 weeks after treatment,and 6 weeks after treatment.The expression levels at 2 weeks and 6 weeks after treatment were significantly lower than that before treatment.The levels of pemphigus antibodies(anti-Desmoglein 1 and anti-Desmoglein 3 antibodies)did not change significantly 2 weeks and 6 weeks after treatment.The relative expression of miR-338-3p is associated with disease severity of pemphigus after adjusting for the baseline characteristics and other clinical parameters.3.Screening target genes of miR-338-3p:1)PBMCs from health controls were successfully isolated and grew as suspended oval cells.2)Upregulation of the expression level of miR-338-3p in PBMCs of normal people can lead to the down-regulation of RNF114 messenger RNA(mRNA)and RNF114 protein expression.3)Luciferase reporter gene suggested that miR-338-3p binded to RNF114 mRNA,which is the target gene for miR-338-3p.4)RNF114 was up-regulated in PBMCs in pemphigus patients at mRNA and protein level.ConclusionMiR-338-3p expression level was significantly elevated in patients with active pemphigus and was not elevated in patients with inactive pemphigus and patients with active BP.The Area Under the Curve(AUC)of ROC curve was 0.8919,suggesting that miR-338-3p can be used as a biological marker of pemphigus activity and diagnosis.The expression level of miR-338-3p was correlated with the severity of the disease and more sensitive than the pemphigus antibody,which can be used as a monitoring indicator of the degree of activity of pemphigus.RNF114 is a target gene of miR-338-3p.The high expression of RNF114 in patients with pemphigus may be explained as:due to the autoimmune state,the expression of RNF114 in patients with pemphigus were up-regulated which could promote T cell activation and autoantibody production.Since miR-338-3p binds to RNF114 and induces its degradation,miR-338-3p in pemphigus patients is upregulated through the negative feedback. |
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