MiRNA Expression Profiling And Function In Angiosarcoma

Angiosarcoma is a mesenchymal cell line-derived malignant tumor.Tumor cells have some normal vascular endothelial morphological and functional characteristics. Angiosarcoma incidence is relatively low, less than2%in soft tissue sarcoma, tumor cells invasive growth, and a high degree of malignancy. Diagnosis of angiosarcoma depends mainly on histopathological methods, due to the lack of diagnostic specificity, it is difficult to diagnosis in difficult cases, easily lead to misdiagnosis; Angiosarcoma taken to surgery combined with chemotherapy, the treatment less effective, the median survival of only1.5years. Therefore, searching for new clinical diagnostic and therapeutic methods have greater clinical significance. Existing studies have shown that abnormal expression of miRNA plays an important role in tumor development, some tumors has been revealed differential expression of miRNA, its potential as a clinical diagnostic, prognostic markers and through its tumor-related target gene research to development of potential targeted therapies, this provides a new way for us to study angiosarcoma. Therefore, we try to profiling angiosarcoma miRNA expression, capillary hemangioma as a benigh control, and predict the difference expression of miRNA target genes and analysis the signaling pathways, also study the related of gene function. This study consists of two parts.. This study is divided into two parts. Part Ⅰ: miRNA expression profiling in angiosarcomasObjective:Screening the different miRNA expression in angiosarcoma,and analysis the tumor related genes and signal pathway to provide basic data for further study.Method:Gathering27cases of angiosarcoma and15cases of capillary hemangiomas paraffin tissue to extraction total RNA,and to study3cases of angiosarcoma and3cases of capillary hemangiomas miRNA expression microarray,choose some different expression miRNA and use qPCR to enlarge the sample verification.,then through the method of bioinformatics analysis of tumor related genes and signal pathway analysis of miRNA screened.ResuIt:Using miRNA microarray to screen out of32differentially expressed miRNA,there are18miRNA which up-regulation and14miRNA down-regulation, The12different expression of miRNA reached the condition of the signal value is greater than500and the difference ratio greater than2,which7miRNA up-regulation and5miRNA down-regulation.by using qPCR to enlarge the sample verification,succeed to screen4differentially expressed miRNA (miR-222-3P, miR-378-3p, miR-483-5p, miR-497-5P),and obtaining a large number of target genes and signaling information through Bioinformatics Method.Part Ⅱ:The expression and role of KCa3.1a target gene of hsa-miR-497-5p in angiosarcomaObjective:Objective:detecting expression of KCa3.1in angiosarcoma,discussing biological functions of KCa3.1in angiosarcoma.Method:Using qPCR to detected the expressing of ISO-HAS tumor associated14potassium of angiosarcoma cell line,using Western blot, IHC to test related protein, then using the whole cell patch clamp for ion channel current detection,studying on the screened potassium ion channel subtype in the proliferation, cycle, invasion and other biological functions.Result:we detected the expression of KCa3.1gene and protein in ISO-HAS cells, at the same time,detected the expression Kca3.1in20cases of angiosarcoma and15cases of capillary hemangiomas tissues using the IHC method, and17cases of angiosarcoma with respect to capillary hemangiomas significantly high expression, the remaining3cases with lower expression just like capillary hemangiomas. Record to Kca3.1current using whole-cell patch clamp recording, the average current size is74pA, further confirmed the expression of Kca3.1.5μ M,10μ M, Kca3.1specific inhibitor TRAM-3420μ M could significantly inhibit the proliferation of ISO-HAS cell, increase the percentage of G0-G1phase, and significantly reduced the invasive; using Kca3.1specific siRNA could significantly reduce the expression of Kca3.1, and inhibit the proliferation of ISO-HAS cells, Increase the percentage of G0-G1phase.Conclusion:We speculate that the miRNA plays an important role in the occurrence and development of angiosarcoma, spectrum analysis by miRNA expression, screening the differential expression of miRNA, And used bioinformatics methods to obtain a large number of prediction of tumor associated genes and pathways,these miRNAs as a potential clinical diagnostic markers;through the available information,we found that Kca3.1plays an important role in a series of the biological function of angiosarcoma.