Building The Regulatory Pathway Of Nr3c2 In Anxious Animal Model By Using System Biology Approach

ObjectiveThis study is attempted to identify new stress and anxiety related genes and understand the molecular mechanism of Nr3c2 involvement in stress and anxiety through construction of Nr3c2 gene network.Methods1. This project focused on the study of gene networks and their regulation mechanism that are involved in the anxiety disorder using the date of gene expression and eQTL from stress and control groups that were produced in previous project and methods of system biology including genetics, bioinformatics, genomics, expression profiling, genotypes, statistics and molecular biology.2. We validated the gene regulating pathway of Nr3c2 and construct gene regulatory networks in vitro using the techniques of cell culture, siRNA and qPCR.3. We validated the gene regulating pathway of Nr3c2 and construct gene regulatory networks in vivo in chronic stress mouse model using Western Blot, qPCR, and immunofluorescence.Results1. System biology methods were used to screen the upstream candidate genes of Nr3c2. Lrp11 and Pex3 were identified as best candidate genes for further validation.2. In order to investigate whether the two upstream candidate genes of Nr3c2 are associated with stress, a model of chronic unpredictable stress was built to detect gene expression change. The result showed the expression of Lrp11 is higher in anxiety group, while the gene expression of Pex3 was decreased, compared with the control group(P<0.05), suggesting that Lrp11 and Pex3 are associated with stress response.3. To verify the upstream regulation pathway of Nr3c2, HEK 293 T cells and SH-SY5 Y neuroblastoma cells were transfected with siRNA duplexes using Lipofectamine 2000. The results shown down-regulation of Pex3 gene led to a significantly decreased expression of Nr3c2(P<0.05), however, down-regulation of Lrp11 gene has no statistically significant effection on Nr3c2 gene expression.Conclusions1. Building the regulatory network of Nr3c2 gene provides a new basis for the molecular mechanism of anxiety research.2. Pex3 is the upstream gene of Nr3c2 and may play an important role in stress response.3. The Lrp11 is new candidate gene involved in stress response, but it is not upstream gene of Nr3c2. Building the regulatory network of Lrp11 gene, provides a new basis for the molecular mechanism of anxiety research.