Analysis Of The Gene Expression Of Rat Brain Affected By Nicotine Through RNA-Seq

ObjectiveThe study applied RNA sequencing (RNA-Seq) to investigate the gene expression profile in prefrontal cortex (PFC), dorsal hippocampus (HIP) and dorsal striatum (STR) regions of HIV-1transgenic (HIV-1Tg) rat’brain and Fischer344(F344) rat’ brain affected by nicotine and explore the effects of nicotine on the gene expression in PFC, HIP and STR regions of HIV-1Tg rat’brain.Methods1. Twenty-four adult male HIV-1Tg rats and twenty-four F344adult male control strain rats were randomly divided into treatment group and control group, with12animals in each group. Animals in the treatment group were injected with nicotine solution while those in the control group were given physiological saline for17days. Thus there were four groups of rats (n=12in each group) were tested:F344Saline, F344Nicotine, HIV-1Saline, and HIV-1Nicotine.2. At the end of drug administration, the tissue of PFC, STR and HIP regions were sliced from the brain of each animal. RNA-Seq was utilized to analyze the gene expression profile in PFC, STR and HIP regions of each animal’s brain, respectively. Bioinformatics tools including Bowtie, Tophat and Cufflinks were used to map the sequencing reads to the reference genome of rat, and to measure the relative expression level of each transcript.3. After measure the relative expression level of each transcript using RPKM method, t test was used to identify the genes whose expression levels were significantly different between the two groups (F344Saline vs F344Nicotine; F344Saline vs HIV-1Saline). Analysis of Variance (ANOVA) was applied to investigate the interaction of strain (HIV-1Tg rat vs F344rat) and effect (nicotine vs saline) and futher t test was used to investigate the effect of nicotine on the gene expression of HIV-1Tg rat. The functional categories and the biochemical pathways associated with these genes were further analyzed by Onto-Express and IPA.Results1. By comparing the expression level of each transcript between F344Saline group and F344Nicotine group,50,50.383significantly differentially expressed genes affected by nicotine in PFC, STR and HIP were identified, respectively. These genes were enriched in multiple Gene Ontology categories (e.g., ion transport, transcriptional regulation and signal transduction) and IPA pathways (e.g., protein kinase A signaling, ERK/MAPK signaling, synaptic long term potentiation and mitochondrial dysfunction).2. By comparing the expression level of each transcript between F344Saline group and HIV-1Saline group,245,385,139significantly differentially expressed genes in PFC, STR and HIP of HIV-1Tg rats were identified, respectively. These genes were enriched in multiple Gene Ontology categories (e.g., immune response, transcriptional regulation, ion transport, neurotransmitter metabolism and DNA damage response) and IPA pathways (e.g., dopamine receptor signaling, Wnt/(3-catenin signaling, apoptosis of target cells, interferon signaling and LXR/RXR activation).3. Through the analysis of variance to the four groups, the interaction of strain and effect was detected. By comparing the expression level of each transcript between F344Saline group and HIV-1Saline group, and between HIV-1Saline group and HIV-1Nicotine group,23,13,11significantly differentially expressed genes restored by nicotine in PFC, STR and HIP of HIV-1Tg rats were identified, respectively, including Rps20, Ndufa4, Fgf9, et al.Conclutions1. By altering the expression of certain genes in PFC, STR and HIP regions of F344rat" brain, nicotine treatment could regulate signal transduction, transcriptional regulation, ion transport and other biological processes, thus could cause the neurological dysfunctions.2. Due to the abnormal expression of related genes in PFC, STR and HIP of HIV-1Tg rat’brain, many biological processes including myelination, Wnt/β-catenin signaling, signal transduction were altered, thus could lead to the damage of the central nervous system.3. Nicotine and HIV-1viral proteins may have opposite effects by regulating their shared pathways in opposite, so nicotine could restore the genes and pathways altered in the HIV-1Tg rats, thus may have neuroprotection effect on the central nervous system of HIV-1infection individuals.