Arthritis & Rheumatism, Volume 62,
November 2010 Abstract Supplement
Abstracts of the American College of
Rheumatology/Association of Rheumatology Health Professionals
Annual Scientific Meeting
Atlanta, Georgia November 6-11, 2010.
Altered microRNA Expression in the Murine Tri-Congenic B6.Sle123 Model.
Garchow1, Barry, Leung3, Yiu Tak, Caricchio2, Roberto, Kiriakidou1, Marianthi
Statement of Purpose:
microRNAs (miRNAs) have emerged over the last decade as a conserved class of non-coding RNAs that regulate gene expression. Current evidence supports a key role for miRNAs in the development and function of the immune system and emerging evidence underscores the importance of this pathway in autoimmunity. However, the expression and function of miRNAs in SLE and the signaling pathways regulated by miRNAs in lupus remain largely unknown.
Research in our laboratory focuses on the function of miRNAs in the tri-congenic mouse model B6.Sle123. Autoimmune disease in B6.Sle123 is characterized by autoantibodies, lymphosplenomegaly and glomerulonephritis, strongly resembling human lupus. We studied miRNA expression in mouse lupus B and T cells over the course of twelve months; before manifestation of renal disease and as it progresses from mild proteinuria to fatal nephrotic syndrome.
Mice (B6.Sle123 and C57BL6/J) were sacrificed at 2, 6 and 12 months of age and their spleens harvested. Splenocytes from individual mice were FACS purified into CD19+, CD3+CD44lowCD62Lhigh and CD3+CD44lowCD62Lhigh cell populations. For quantitative real-time PCR, total RNA from purified cells was reverse-transcribed using microRNA specific stem-loop primers. RT products were amplified by real-time PCR. Fold expression differences were calculated using the DDCt method. For Northern blotting, total RNA from purified cells was separated on 15% urea-polyacrylamide gels, blotted and UV crosslinked. microRNAs were visualized by autoradiography and quantitated using the GelQuant software. For Western blotting, total protein from purified cells were separated by PAGE and blotted on nitrocellulose membranes. Bands were visualized by chemiluminescence and quantitated with ImageJ software.
We asked if altered microRNA expression in B6.Sle123 B and T lymphocytes correlates severity of renal disease. Here we report that several microRNAs are differentially expressed in B and T lymphocytes from lupus mice and their expression correlates with the severity of lupus nephritis. All eight miRNAs that we showed as upregulated in B cells from mice with severe SLE nephritis (miR-21, 34a, 221, 222, 223, 155 and 142-5p) and have been also recently reported as upregulated in human lupus nephritis.
Our findings show that several microRNAs with known key roles in the development and function of lymphocytes are differentially expressed in B6.Sle123 derived B and T cells and their expression correlates with the severity of renal disease. Correlation of our results with recently published data of miRNA expression in PBMCs from patients with lupus nephritis indicates that B6.Sle123 is a suitable model to study the miRNA function in lupus. We are now focusing on identifying miRNA-dependent signaling pathways that are uniquely affected in this model, in which disease manifestations and disordered mechanisms overlap with those in human SLE. In our studies we employ novel in vivo experimental methods to knock down endogenous miRNAs in B and T cells in order to dissect these pathways.
To cite this abstract, please use the following information:
Garchow, Barry, Leung, Yiu Tak, Caricchio, Roberto, Kiriakidou, Marianthi; Altered microRNA Expression in the Murine Tri-Congenic B6.Sle123 Model. [abstract]. Arthritis Rheum 2010;62 Suppl 10 :422