Screen Differentially Expressed MicroRNAs In Peripheral Blood Celts Of Idiopathic Thrombocytopenic Purpura Patients

Objective：Screening microRNAs in peripheral blood cells of initially treated idiopathicthrombocytopenic purpura patients and controls and analysising differentially expressedmiRNAs between idiopathic thrombocytopenic purpura patients and controls to providetheory and practice basis for new idiopathic thrombocytopenic purpura gene diagnosis,prognostic evaluation system and new molecular therapy targets.Methods：Extract3ml venous blood that EDTA anticoagulation of5idiopathicthrombocytopenic purpura patients and5controls,collect peripheral blood mononuclear cellsby1×RCLB,make sure that the number of white blood cells more than106in every sample.Total RNA was isolated from every sample using TRIzol (Invitrogen) and miRNeasy minikit (QIAGEN) according to manufacturer’s instructions. RNA quality and quantity wasmeasured by using nanodrop spectrophotometer (ND-1000, Nanodrop Technologies) andRNA Integrity was determined by gel electrophoresis. After RNA isolation from the samples,the miRCURY Hy3/Hy5Power labeling kit (Exiqon, Vedbaek, Denmark) was usedaccording to the manufacturer’s guideline for miRNA labelling. After stopping the labelingprocedure, the Hy3TM-labeled samples that mixture of two groups were hybridized on themiRCURYTMLNA Array (v.16.0)(Exiqon) according to array manual. Then the slides werescanned using the Axon GenePix4000B microarray scanner (Axon Instruments, Foster City,CA). Scanned images were then imported into GenePix Pro6.0software (Axon) for gridalignment and data extraction. Replicated miRNAs were averaged and miRNAs thatintensities>=50in all samples were chosen for calculating the Median normalization.Hierarchical clustering was performed using MEV software (v4.6, TIGR). Differentiallyexpressed miRNAs were analysised between idiopathic thrombocytopenic purpura patientsand controls. The results were checked by using RT-PCR.Results：There were159differential expression of which79up-regulated and80down-regulated in idiopathic thrombocytopenic purpura.Three miRNAs were checked by usingRT-PCR,miR-31was significant up-regulated,miR-181a and miR-148a were significantdown-regulated.The results after checking by RT-PCR were coincided with microarrayanalysis. Conclusions：1.MiRNAs were screened from ITP patients by using high-throughput microarraytechnology, there were159differential expression of which79up-regulated and80down-regulated in idiopathic thrombocytopenic purpura. The results after checking byRT-PCR were coincided with microarray analysis to demonstrate that the microarraytechnology was reliable and exact.2.Try to establish biomarkers index of ITP diagnosis according to the model ofdifferentially expressed miRNAs and set up the specific biologic diagnosis system of ITPfrom genome level.3.We may find new regulatory genes that make connection to immune regulationand thrombopoiesis from differentially expressed miRNAs.4.To provide the basis of new molecular therapeutic targets for the treatment of ITP.