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Investigation of Relationship between Cytokines Gene Expression and miRNA in Systemic Lupus Erythematosus Patients
miRNAs transpire as promising elements in molecular medicine for the identification of new diagnostic, prognostic and targeting therapeutic biomarkers. This being the case, we aimed to investigate a or a group of specific diagnostic biomarkers for Systemic Lupus Erythematosus (SLE) disease, to identify cytokines genes’ expression profiling, comparison between the profiles with related amounts in the serum and eventually to study target-gene-mediated functional roles of miRNAs, which have been correlated to disease development and progression.
First, blood and serum samples were obtained from a total of 24 volunteers, including 16 patients (10 with renal involvement and 6 without renal involvement), and eight healthy controls. In order to use in microarray assays total RNA and miRNAs samples that were isolated from PBMCs and serum samples respectively, were assessed by using 8x60K v2 “SurePrint G3 Human Gene Expression” and 8x60K v19 miRNA microarray slides (Agilent). Data processing and differential expression analysis were done by "GeneSpring" software (version 12.6) (Agilent). Taking coexistence of factors such as the renal involvement, complement deficiency, positive ANA and anti-DNA, into the account, miRNA and mRNA microarray analysis, was performed by comparison of SLE patients with the healthy controls. For each differentially expressed miRNA, potential target genes were predicted by microRNAorg, TargetScan and PITA prediction tools. Obtained mRNA profiling data were interrogated for the target genes and mRNA/miRNA binding site sequence analyses were done. Finally, the amounts of cytokines were measured by multiplex ELISA method (Bio-Rad).
The results of study showed that some differentially expressed miRNAs may play pivotal roles in PI3K-AKT-mTOR pathway. These included (hsa-miR-1825, hsa-miR-933 and hsa-miR-149-5p) of the 10 differentially expressed miRNAs in SLE ​​patients comparing to healthy controls, hsa-miR-766-3p from those which were differentially expressed in SLE patients with renal compared to without renal involvement, and hsa-miR-621 among those which were differentially expressed in SLE without renal involvement and positive ANA test compared to the others. In addition, according to the obtained data it is suggested that blood-borne proinflammatory cytokines such as IL-4, IL-6 and TNF-α alongside with stage and severity of disease may contribute to differential expression of these miRNAs which may also lead to much more serious complications such as nephropathy, hypertension, dark spots on the skin, and insulin resistance.
Furthermore, out of the target genes predicted for hsa-miR-149-5p through bioinformatics studies, ERbB3 was confirmed experimentally in the mRNA expression analysis. Based on NCBI sequence matching analysis results, with approximately 90% homology level, it was shown that hsa-miR-149-5p regulates gene expression via degradation of primer transcript prior to mRNA splicing.
Because of the smaller sample size and clinical heterogeneity of SLE, our results must be confirmed in larger studies.

  

ŞAHİN Kaniye
Danışman : Prof. Dr. Nermin GÖZÜKIRMIZI

Anabilim Dalı : Moleküler Biyoloji ve Genetik

Programı : -

Mezuniyet Yılı : 2014

Tez Savunma Jürisi : Prof. Dr. Nermin GÖZÜKIRMIZI

Prof. Dr. Şule ARI

Prof. Dr. Ayhan DEVİREN

Doç. Dr. Ahu ALTINKUT UNCUOĞLU

Doç. Dr. İbrahim İlker ÖZYİĞİT