Study On Gene Expression And Function Of Human And Mouse Brain Transcriptome Gene And Synergistic Interaction Between Dopamine D5 Receptor And Gastrin Receptor In Urinary Sodium Excretion

Comparative analysis of gene expression and function between human and mouse brain tissuesBackground:Neurodegenerative disorders have become serious threatens to human health and quality of life along with the aging population. Up to present, there are no effective drugs to prevent the progression of such disease clinically. It is of fundamental importance to design an optimal animal model to explore the underlying mechanisms and screen drugs. Since mouse has a small size, a short gestation period, a rich experimental history, as well as a mature genetic engineering technology, it has been extensively used as a tool for investigating the onset and development of human neurodegenerative disorders. As a first step to construct transgenic mouse model of human brain lesions, it is significantly important to clarify the similarity and divergence of genetic background between non-diseased human and mouse brain tissues. Therefore, understanding the similarities and differences between human and mouse corresponding brain regions, to some extent, may provide overall framework to establish mouse models for neurodegenerative disorders and evaluate their effectiveness and accuracy. Meanwhile, it can also contribute to comprehending the underlying causes of the discrepancy in learning, memory and cognition across human and mouse.Methods:Microarray chip datasets from healthy adult human and mouse PFC, HIP and STR were downloaded from the GEO database. Preprocessing of microarray data was performed using Expression Console software. The robust multichip average (RMA) algorithm was selected for signal intensity normalization. Percentile method was used to mine the highly expressed genes and functional enrichment analysis was performed using DAVID. Venn analysis was used to depict the overlapping ratios of the notably enriched biological process (BP) terms between two brain tissues. GOSemSim, an R package, was selected to perform GO semantic similarity analysis. After othologs detection and signal adjusting, the expression divergence was calculated using one minus Pearson’s Correlation Coefficient. Clustering analysis was performed using both hierarchical clustering algorithm and principal component analysis. Lineage specific expressed orthologous genes were identified by comparison of the most extreme sub-datasets across species and further verified using experiments. Compared to mouse, differentially expressed orthologous genes from human brain tissues were identified by combining limma algorithm with fold change of percentile (FCP). Gene-gene interactions were detected using String database and visualized using Cytoscape software. We then analyzed the topological properties of the network and used the MCODE algorithm to mine the highly connected modules. DAVID was performed to analyze the biological functions of the identified modules. The miRNome expressions in human and mouse hippocampus were detected using next-generation sequencing technology. After preprocessing of the raw data, the known mature miRNAs, other small RNAs and novel miRNAs were determined. TargetScan algorithm was selected to predict target mRNAs. DAVID software was used to perform functional enrichment analysis for target genes. Expression levels of mature miRNAs were normalized by transcripts per million (TPM). The functional roles of hippocampus-enriched or species-specific expressed miRNAs were determined by DIANA-mirPath v3.0. We then used Spearman correlation coefficient to analyze the expression pattern across species. Differentially expressed miRNAs between human and mouse hippocampus were detected by two criterion:edgeR algorithm and fold change. The regulatory relationships between differentially expressed miRNAs and target mRNAs were considered as the combination of all predicted targets from four major algorithms: miRanda、Pictar、DIANA-microT and Targetscan. Furthermore, according to the principle of retroegulation, we established a relative method to indirectly screen the miRNA-mRNA interacting pairs. The functional miRNA-mRNA regulatory network was visualized using Cytoscape software and the biological functions were determined by the roles of target mRNAs in the network using DAVID software.Results:The dominating enriched BPs of the highly expressed genes in brain tissues were very similar across species. The enriched KEGG pathways and their statistical significances of the highly expressed genes from human and mouse brain regions were similar. The expression pattern of the orthologous genes were highly conserved across human and mouse PFC、HIP and STR. However, the number of functionally enriched BP terms of the highly expressed genes in human brain tissues was approximately two-fold than that of mouse corresponding brain tissues; the number of BP terms associated with neural activities was evidently more than that of human, among which neuron projection morphogenesis and development was human specific. The significances of common enriched BP terms were clustered relying on species. Hierarchical clustering analysis highlighted a more species-dominated consistency in biological function. COX5B, WIF1, SLC4A10 and PLA2G7 were identified as species-specific expressed orthologous genes. Compared with mouse brain areas, human corresponding DEHGNs had both small world and scale-free features, and there existed functional sub-network modules associated with neural activities. We identified a novel miRNA, termed novel-21-5p, whose most target genes were expressed in synapse, synaptosome, vesicle, postsynaptic density and projection neuron, which was involved in neurotransmitter, neurotrophin signaling pathway and glioma. miR-9-5p is the most enriched miRNAs in both human and mouse hippocampus. The first 20 enriched miRNAs in human hippocampus were significantly associated with multiple neuro-related biological processes such as neurotrophin TRK receptor signaling pathway, synaptic transmission and axon guidance. Furthermore, compared to mouse hippocampus, a human hippocampus specific expressed miRNA, namely miR-656-3p, was involved in the biological processes of synaptic transmission, neurotrophin TRK receptor signaling pathway and neuron cell-cell adhesion. The expression pattern of orthologous miRNAs between human and mouse hippocampus was evidently conserved and the expression conservation was positively correlated with the sequence conservation. The differential miRNA-mRNA regulatory network were established according to a reverse regulatory relationship between computational predictions and differentially expressed miRNAs. The down-regulated network were notably associated with pattern specification process, regionalization, anterior/posterior pattern formation, visual perception and sensory perception of light stimulus.Conclusion:Our study shows that the biological functions of the highly expressed genes in brain tissues are similar between human and mouse; highly enriched miRNAs in human and mouse hippocampus are similar; the expression pattern of brain orthologous mRNAs and miRNAs are highly conserved across species. Our study can provide the genetic basis for understanding the high concordance of anatomical structure and cell type across human and mouse brain tissues. In addition, to some extent, mouse can be effectively and accurately used for modeling the neurophysiological and pathologic processes. However, the biological functions of the highly expressed genes in human brain are more diverse and intricate than mouse; and the biological functions tend to be a more species-dominated similarity; and there exist functional sub-networks related with neural activities between human and moue corresponding brain tissues; the novel, species specific expressed, highly enriched and the down-regulated miRNA-miRNA functional regulatory network are involved in important neuro-related biological functions, providing a genetic basis for comprehending the discrepancy in learning, memory and cognition between human and mouse.The synergistic roles of cholecystokinin B and dopamine D5 receptors on the regulation of renal sodium excretionBackground:Renal dopamine D1-like receptors (D1R and D5R) and the gastrin receptor (CCKBR) are involved in the maintenance of sodium homeostasis. The D1R has been found to interact synergistically with CCKBR in renal proximal tubule (RPT) cells to promote natriuresis and diuresis. D5R, which has a higher affinity for dopamine than D1R, has some constitutive activity. Hence, we sought to investigate the interaction between D5R and CCKBR in the regulation of renal sodium excretion.Methods:We used immunoblotting to measure D5R protein expression in HK-2 cells treated with gastrin (a CCKBR ligand), YF476 (a selective CCKBR antagonist), gastrin coupled with YF476, respectively; and measure CCKBR protein expression in HK-2 cells treated with fenoldopam (a selective D1R/D5R agonist), Sch23390 (a selective D1R/D5R antagonist), fenoldopam coupled with Sch23390, respectively; and also test the concentration- and time-dependent effects corresponding to gastrin induced D5R expression or fenoldopam induced CCKBR expression. The specificity of the interaction between D5R and CCKBR was further verified in HEK293 cells heterologously expressing both human D5R and CCKBR (HEK293-D5R-CCKBR) andRPT cells from a male normotensive human (NT). We next performed co-immunoprecipitation and co-localization experiments to test whether the interaction between D5R and CCKBR was direct or not in HK-2 and HEK293-D5R-CCKBR cells and BALB/c mouse RPTs. Renal D5R or CCKBR protein expression in CCKBR-/- or D5R-/- mice was also detected using immunoblotting.4-month old male BALB/c mice were fed high-salt diet for two weeks, and separated into seven groups, and intraperitoneally injected with vehicle (normal saline), Sch23390, YF476, fenoldopam, gastrin, fenoldopam coupled with YF476 and gastrin coupled with Sch23390, respectively, daily for one week. At the end of the drug treatment, urine was collected for 24 hours, and urine sodium and creatinine concentrations were measured.Results:We found D5R and CCKBR increase each other’s expression in a concentration- and time-dependent manner in HK-2 cells, the specificity of which was verified in HEK293 cells heterologously expressing both human D5R and CCKBR and in RPT cells from a male normotensive human. Also, D5R and CCKBR colocalized and co-immunoprecipitated in BALB/c mouse RPTs and human RPT cells. CCKBR protein expression in plasma membrane-enriched fractions of renal cortex (PMFs) was greater in D5R-/- mice than D5R+/+ littermates and D5R protein expression in PMFs was also greater in CCKBR-/- mice than CCKBR+/+ littermates. High salt diet, relative to normal salt diet, increased the expressions of CCKBR and D5R in PMFs. Disruption of CCKBR in mice caused hypertension and decreased sodium excretion. The natriuresis in salt-loaded BALB/c mice was decreased by YF476, a CCKBR antagonist and Sch23390, a D1R/D5R antagonist. Furthermore, the natriuresis caused by gastrin was blocked by Sch23390, a selective D1R/D5R antagonist, while the natriuresis caused by fenoldopam, a D1R/D5R agonist, was blocked by YF476, a selective CCKBR antagonist.Conclusion:Our findings indicate that CCKBR and D5R synergistically interact in the kidney, which may contribute to the maintenance of normal sodium balance following an increase in sodium intake.