Studying the Role of Apoptosis-Related Long Non-Coding Rnas in Systemic Lupus Erythematosus by Applying Mouse Models, Patient Samples and Crispr/Dcas9 Interference Genome Editing Tool

Project: Research project

Project Details


Systemic lupus erythematosus (SLE) is a chronic autoimmune disease with the key pathogenic mechanism as an imbalance between apoptotic cell production and disposal of apoptotic material, resulting in the release of nuclear autoantigens with immune complex (IC) formation and leading to multi-organic inflammation A better understanding of the pathogenesis is needed to develop novel targeted therapy other than the conventional immunosuppressive agents with higher failure rates and significant adverse effects. To inhibit the apoptotic process through controlling the lncRNAs expression, can not only reduce nuclear autoantigens production and avoid IC formation, but also suppress the apoptosis of phagocytes with restoring the clearance of apoptotic materials. The preliminary experimental results demonstrated up-regulated lincRNA-p21 expression in SLE patients correlated with the disease activity and lupus nephritis (LN) manifestations, and higher lincRNA-p21 expression was found in those with pulmonary hemorrhage (PH). In addition, the doxorubicin-induced Jurkat cell apoptosis was associated with up-regulated lincRNA-p21 and down-regulated competing endogenous (ce) miRNA-181a and target protein IL-2 expression. Furthermore, the pristine-induced Balb/c and C57BL/6 lupus mouse models were established with the presentations of LN and PH, respectively. This research project is aiming at studying the role of apoptosis-related lncRNAs in SLE by applying mouse models, clinical samples and CRISPR/dCas9 interference genome editing tool. In the first year, we will identify dysregulated apoptosis-related lncRNA profiles by screening peripheral mononuclear cells (MNCs) from patients by lncRNA microarray. The MNCs subpopulations and paired urine/blood samples will be analyzed by quantitative real time PCR for selected lncRNAs. The potential ce-miRNA targets and miRNA-related target proteins will be analyzed by luciferase reporter assay and immunoblot/ELISA to elucidate the pathogenic lincRNAs-miRNAs-target proteins axis. In the second year, the spleen, kidney and lung tissues will be examined for the lncRNAs expression from experimental lupus models in Balb/c and C57BL/6 mice. The up-regulated lncRNAs related to pro-apoptosis process identified in both patients and mice with similar kinetic profiles will be the target lncRNA candidates in addition to lincRNA-p21. We will prepare the CRISPR/dCas9 with KARA or KRAB-MeCP2 repressor domain containing designed sgRNAs targeting lincRNA-p21 and other candidate lncRNAs, and transfect RAW 264.7 mouse and Jurkat human cells to create stable transfectants for further real-time PCR screening. The transfected cells will be subjected to apoptosis analyses. In the third year, we will apply the in vivo knockdown of lncRNAs in lupus mouse models to elucidate the roles of these molecules in LN and PH manifestations through intravenous administration of CRISPR interference targeting lincRNA-p21 or other candidate lncRNAs in pristine-injected Balb/c and C57BL/6 mice, respectively. The efficacy of lncRNA knockdown will be examined by real-time PCR and in situ hybridization, and the evaluation of LN and PH will be performed by proteinuria amounts and hemoglobulin levels, respectively, as well as histopathological analyses. Expected results from this project through a proof-of-concept approach will contribute to elucidate the pathogenic role of lncRNAs in SLE and as potential therapeutic targets in such patients.
Effective start/end date22-08-0123-07-31


Explore the research topics touched on by this project. These labels are generated based on the underlying awards/grants. Together they form a unique fingerprint.