Genetic Analysis E2F-associated Phosphoprotein(EAPP) Gene Regulatory Networks In Stress-induced Anxious Model

ObjectiveThe purpose of this project is to study the gene expression change and regulationof EAPP gene in mouse hypothalamus during chronic stress. Methods1. The expression change of EAPP during chronic stress was tested with western blot on protein level and with qPCR(quantative polymerase chain reaction) on the mRNA level. The distribution and expression level of EAPP in hypothalamus was identified by immunohistochemical staining.2. eQTL mapping of EAPP was performed through Genenetwork Website(www.genenetwork.org) using tools of the interval mapping,composite mapping as well as interactive mapping. This study used the date set of ―INIA BXD hypothalamus Cohort AffyMoGene 1.0 ST(Nov10) RMA‖(accession number: GN113) in the Genenetwork Website(www.genenetwork.org). This data contains gene expression from 50 BXD RI strains, 2 reciprocal F1 hybrids, and the B6 and D2 parental strains.3. The downstream candidate genes of EAPP were identified by genetic correlatin,cluster map and partial correlation analysis.4. The validation of downstream genes and high-associated genes of EAPP. After knockdown of EAPP, the expression changes of downstream genes–Sphk2 and Nosip gene as well as high-associated genes–Mmp9,Npy,Npy5 r and Maob were tested in SH-SY5 Y cells using qPCR. The expression change of Sphk2 and Nosip as well as was Mmp9,Npy,Npy5 r and Maob also detected in chronic stress model by qPCR method.5. Build EAPP regulatory networks. Pearson’s correlation coefficient(person product-moment correlation coefficient) as an evaluation index was used to calculate the genetic correlation between the expression of EAPP and all other genes in the hypothalamus of BXD RI mice. Result1. In the hypothalamus of chronic stress mouse, EAPP rise at both the mRNAlevel detected by real-time PCR and the protein level, detected by Western blot. Thisresult is also confirmed in tissue level by immunohistochemical analysis. Theseresults suggested that EAPP is involved in stress response.2. eQTL mapping showed that EAPP has significant cis-e QTL with thelikelihood ratio(LRS) of 100(P < 0.01).3. Sphk2,Kih132 and Nosip genes were identified as the downstream genesof EAPP. In the chronic stress model,the Sphk2 and Nosip mRNA showed highexpression. Sphk2 and Nosip genes mRNA showed low expression followingknockdown of EAPP in SH-SY5 Y cells.4. The gene network of EAPP has been built through genetic correlatinanalysis, and Mmp9,Npy,Npy5 r and Maob are confirmed as member of EAPPgene network through qPCR. Conclusion1. The expression of EAPP increased during chronic stress and exhibited morehigh correlation with the stressed phenotype2. The EAPP gene is a cis-eQTL and can regulated the stress related genes, suchas:Npy,Maob,Npy5 r,Mmp93. EAPP genetic regulatory network was built...