| Objective To detect the expression of five kinds of small RNA(micro RNA, mi RNA) from patients with Ankylosing Spondylitis, investigating the differences among those micrornas. And then to further explore the relevant between those micornas with the pathogenesis of Ankylosing Spondylitis, To investigate whether mi RNAs can serve patients with ankylosing spondylitis potential diagnostic markers, and to study the regulation of mi R-495 target to the protein of DVL-2.Method 1. Based on the literatures, find out the closely relevent genes to the AS, and then to determine the closely relevant mi RNAs with the AS through the online bioinformatics database. By Real-time PCR method to detect the expression of mi R-17, mi R-106, mi R-181, mi R-495 and mi R-30 in 61 cases of AS patients and 31 cases of healthy peripheral blood mononuclear cells.At the same time by using the statistical software SPSS13.0 to analyze the experimental results and filtering the mi RNAs which can be a potential diagnostic biomarkers of AS.2. Combine the literatures with the software tools mi Randa, Targetscan and bioinformatics to predict the genes such as mi R- 495 which has significant difference expression in patients of AS peripheral blood mononuclear cells as a potential target gene. By dual-luciferase reporter gene system and Western blot experiments to verify the relationship between mi R-495 and the target gene of DVL-2.3. Application of ELISA to detect the DVL-2 protein level in plasma of 28 patients with AS and 14 healthy persons. And study the relationship between the expression of mi R- 495 in AS patients’ peripheral mononuclear cells with DVL- 2 protein levels in plasma and mi R-495 by targeting inhibition of DVL- 2 protein to regulate the pathogenesis of ASResults 1. Through the detection clinical samples of 92 cases showed: Compared with normal subjects, upregulation of mi R-106 and have mi R-30 in peripheral blood of patients with AS, but the difference was not significant; downregulation of mi R-181(p<0.05), mi R-495(p<0.05) and mi R-17. By ROC curve analysis the expression of mi R-181 and mi R-495 were combined with clinical diagnosis, the area under the mi R-181 curve was 0.697, sensitivity was 76.67%, specificity of 54.84%, accuracy is 68.48%.hrough the analysis of the results, we believe that the expression level of mi R-181 which has a certain significance in clinical diagnosis of AS disease, but it has relatively low accuracy, so it has low value for the reference of clinical diagnosis.And the area under the curve of mi R-495 was 0.729, sensitivity of 100%, specificity of 58.06%, accuracy is 86.9%. The results show that the expression levels of mi R-495 which provides the reference value in AS clinical disease diagnosis, is expected to become the ankylosing spondylitis disease potential diagnostic markers.2. The bioinformatics analysis results show that mi R-495 can be target to DVL-2 gene.Dual luciferase reporter results showed: the relative luciferase activity of DVL-2 was inhibited by overexpression mi R-495;rather inhibit the expression of mi R-495 promotes that the activity relative fluorescence of DVL-2..3. Further clinical samples to detect the expression of DVL-2 in plasma by ELISA method, compared with healthy control group, the expression of DVL-2 increased in the group of AS. Correlation analysis was conducted between the expression of DVL-2 protein in plasma and the expression of mi R-495 in peripheral blood mononuclear cells.Conclusions 1. The expression level of mi R-495 1 in peripheral blood mononuclear cells, has the reference significance in clinical diagnosis of AS disease, is expected to become the ankylosing spondylitis disease potential diagnostic markers.2. mi R-495 can be targeted regulation the expression of Wnt pathway in the key protein DVL-2, play a regulatory role in AS. |
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