The Differential Expression Of Serum MiRNA In Henoch-Sch(o|¨)nlein Purpura And Henoch-Sch(o|¨)nlein Purpura Nephritis

Henoch–Sch nlein purpura（HSP）is one of the most common forms ofsystemic vasculitis in childhood, which is easy to develop nephritis（HSPN） within6weeks with hematuria and(or) proteinuria. So far, the pathogenetic mechanism ofthe disease has not been understood. Renal biopsy is the gold standard to diagnosekidney disease. However, considering it has traumatic and some risk, not easily beenaccepted by patients, it even cannot be carry out to repeated renal biopsies todynamic judge the effect of treatment and prognosis. So, searching for convenientand effective biomarkers without invasive will be very beneficial to diagnosis andtreatment the disease.MicroRNAs (miRNAs) are endogenous noncoding RNA molecules of18–24nt.Its expression has tissue-cell specific,development temporality and evolutionconservation. It can regulate the expression of target genes in a post-transcriptionalmanner. Evidence indicates that miRNAs play essential roles in pathological andphysiological process, inclulding cell proliferation, differentiation, apoptosis, fatmetabolism, oxidative stress, human diseases formation and so on.Many evidences demonstrate that miRNAs are present in human serum in aremarkable stable form that is resistant to endogenous RNase activity. Furthermore,serum miRNAs are less affected after being subjected to severe conditions(such ashigh temperature,very low or high pH levels and10freeze-thaw cycles). So they cansever as novel minimally invasive biomarkers in diagnosing and monitoring humandisease. Our research aims to use microarray platform to screen significantly differentially expressed miRNAs from serum samples between HSP and HSPN.Then, we applied bioinformatics to predict miRNA targets and explored the roles ofmiRNA played in the disease, laying the foundation for the future clinicalapplication.Materials and Methods1. Screening10serum samples were used to chip experiment from healthycontrols, HSP and HSPN separately.2. Total RNA was extracted from serum using miRNeasy Mini Kit（Qiagen）.The concentration of all total RNA samples were measured byNanoDrop1000(Nanodrop, USA).3. AB TaqMan Human MicroRNA Array(TLDA low-density chip) was usedto screen miRNA(Capitalbio technology company supplied).ApplyingDataAssist2.0software to analyze data to search for differentiallyexpressed miRNAs.4. The microarray results were validated by reverse transcription andquantitative real time PCR.5. MiRNA-targets were predicted through TargetScan, PicTar and miRandasoftwares and cross-referenced with GO (gene ontology)overrepresentation and KEGG pathway analyses.Results1. The extracted total RNA was suited to chip experiment.2. Using2-fold expression difference as a threshold, comparing HSP withcontrol, we identified96miRNAs significantly differently expressed, including7miRNAs up-regulated and89miRNAs down-regulated;comparing HSPN with control, we identified68miRNAs significantlydifferently expressed,including11miRNAs up-regulated and57miRNAsdown-regulated; comparing HSPN with HSP, we identified53miRNAssignificantly differently expressed, including36miRNAs up-regulated and17miRNAs down-regulated.3. RT-PCR was used to validate hsa-miR-15b and hsa-let-7d, which weredifferently expressed in HSP and HSPN. The results demonstrated thatthese miRNAs expression are in accordance with the microarray.4. Using TargetScan、PicTar、miRanda software to predict targets genes of thedifferentially expressed miRNAs, we found there were47miRNAs with617intersection target genes in HSP/NC and HSPN/NC. And there were19miRNAs with435intersection target genes in HSPN/HSP. Then, thesetarget genes were performed GO term analysis,224and219terms wereenriched from these target gene annotations, respectively (p<0.05), relatedto metabolic processes, growth and development, expression andregulations. KEGG pathway analyses showed that these target genes wereenriched in13and11pathways separately (p<0.05), participating in thecell division cycle, cancer development and signaling pathway and so on.Conclusion1. We identified96miRNAs and68miRNAs which were significantlydifferentially expressed in HSP and HSPN separately, also53miRNAs inHSPN/HSP, demonstrating that miRNAs are expressed differently in HSPand HSPN.2. RT-PCR was used to validate2selected miRNAs （hsa-miR-15b、 hsa-let-7d）and the results were in accordance with the microarray.3. The predicted targets of differentially expressed miRNA are involved incell differentiation and proliferation, signaling pathway and transcriptionalregulation. So we consider miRNAs play important roles in HSP and HSPNand can be used as novel biological marker about the disease.