ChIP-seq Profiling Identifies Histone Deacetylase 2 Targeting Genes Involved in Immune and Inflammatory Regulation Induced by Calcitonin Gene-Related Peptide in Microglial Cells.

Calcitonin gene-related peptide (CGRP) is a mediator of microglial activation at the transcriptional level. The involvement of the epigenetic mechanism in this process is largely undefined. Histone deacetylase (HDAC)1/2 are considered important epigenetic regulators of gene expression in activated microglia. In this study, we examined the effect of CGRP on HDAC2-mediated gene transcription in microglial cells through the chromatin immunoprecipitation sequencing (ChIP-seq) method. Immunofluorescence analysis showed that mouse microglial cells (BV2) expressed CGRP receptor components. Treatment of microglia with CGRP increased HDAC2 protein expression. ChIP-seq data indicated that CGRP remarkably altered promoter enrichments of HDAC2 in microglial cells. We identified 1271 gene promoters, whose HDAC2 enrichments are significantly altered in microglia after CGRP treatment, including 1181 upregulating genes and 90 downregulating genes. Bioinformatics analyses showed that HDAC2-enriched genes were mainly associated with immune- and inflammation-related pathways, such as nitric oxide synthase (NOS) biosynthetic process, retinoic acid-inducible gene- (RIG-) like receptor signaling pathway, and nuclear factor kappa B (NF-κB) signaling pathway. The expression of these key pathways (NOS, RIG-I, and NF-κB) were further verified by Western blot. Taken together, our findings suggest that genes with differential HDAC2 enrichments induced by CGRP function in diverse cellular pathways and many are involved in immune and inflammatory responses.

Quantitative Proteomics Identify the Possible Tumor Suppressive Role of Protease-Activated Receptor-4 in Esophageal Squamous Cell Carcinoma Cells.

Exposure to carcinogens of tobacco smoke may result in methylation of protease-activated receptors-4 (PAR4) gene and further induces the loss of PAR4 expression, which is considered to be involved in carcinogenesis of esophageal squamous cell carcinoma (ESCC). Here we employed a TMT-based quantitative proteomic approach to identify PAR4-regulated changes of proteomic profiles in ESCC cells and to identify potentially therapeutic value. A total of 33 proteins were found significantly changed with 15 up-regulated and 18 down-regulated in PAR4-activating peptide (PAR4-AP) treated ESCC cells compared with controls. Bioinformatics analysis showed that key higher expressed proteins included those associated with apoptosis and tumor suppressor (e.g. CASP9), and lower expressed proteins included those associated with anti-apoptosis, autophagy and promoting cell proliferation (e.g. CHMP1B, PURA, PARG and HIST1H2AH). Western blot verified changes in five representative proteins including CASP9, CHMP1B, PURA, PARG and HIST1H2AH. Immunohistochemistry analysis showed that CHMP1B, PURA, PARG and HIST1H2AH expression in ESCC tissues were significantly higher than those in adjacent nontumorous tissues. Our findings will be helpful in further investigations into the functions and molecular mechanisms of PAR4 in ESCC.

Activation of PAR4 Upregulates p16 through Inhibition of DNMT1 and HDAC2 Expression via MAPK Signals in Esophageal Squamous Cell Carcinoma Cells.

A previous study showed that a downexpression of protease-activated receptor 4 (PAR4) is associated with the development of esophageal squamous cell carcinoma (ESCC). In this study, we explored the relationship between PAR4 activation and the expression of p16, and elucidated the underlying mechanisms in PAR4 inducing the tumor suppressor role in ESCC. ESCC cell lines (EC109 and TE-1) were treated with PAR4-activating peptide (PAR4-AP). Immunohistochemistry for DNA methyltransferase 1 (DNMT1) and histone deacetylase 2 (HDAC2) was performed in 26 cases of ESCC tissues. We found that DNMT1 and HDAC2 immunoreactivities in ESCC were significantly higher than those in adjacent noncancerous tissues. PAR4 activation could suppress DNMT1 and HDAC2, as well as increase p16 expressions, whereas silencing PAR4 dramatically increased HDAC2 and DNMT1, as well as reduced p16 expressions. Importantly, the chromatin immunoprecipitation-PCR (ChIP-PCR) data indicated that treatment of ESCC cells with PAR4-AP remarkably suppressed DNMT1 and HDAC2 enrichments on the p16 promoter. Furthermore, we demonstrated that activation of PAR4 resulted in an increase of p38/ERK phosphorylation and activators for p38/ERK enhanced the effect of PAR4 activation on HDAC2, DNMT1, and p16 expressions, whereas p38/ERK inhibitors reversed these effects. Moreover, we found that activation of PAR4 in ESCC cells significantly inhibited cell proliferation and induced apoptosis. These findings suggest that PAR4 plays a potential tumor suppressor role in ESCC cells and represents a potential therapeutic target of this disease.

Analyses of gene expression profiles in the rat dorsal horn of the spinal cord using RNA sequencing in chronic constriction injury rats.

BACKGROUND: Neuropathic pain is caused by damage to the nervous system, resulting in aberrant pain, which is associated with gene expression changes in the sensory pathway. However, the molecular mechanisms are not fully understood. METHODS: Wistar rats were employed for the establishment of the chronic constriction injury (CCI) models. Using the Illumina HiSeq 4000 platform, we examined differentially expressed genes (DEGs) in the rat dorsal horn by RNA sequencing (RNA-seq) between CCI and control groups. Then, enrichment analyses were performed for these DEGs using Gene Ontology (GO) function, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, Hierarchical Cluster, and protein-protein interaction (PPI) network. RESULTS: A total of 63 DEGs were found significantly changed with 56 upregulated (e.g., Cxcl13, C1qc, Fcgr3a) and 7 downregulated (e.g., Dusp1) at 14 days after CCI. Quantitative reverse-transcribed PCR (qRT-PCR) verified changes in 13 randomly selected DEGs. GO and KEGG biological pathway analyses showed that the upregulated DEGs were mostly enriched in immune response-related biological processes, as well as 14 immune- and inflammation-related pathways. The downregulated DEGs were enriched in inactivation of mitogen-activated protein kinase (MAPK) activity. PPI network analysis showed that Cd68, C1qc, C1qa, Laptm5, and Fcgr3a were crucial nodes with high connectivity degrees. Most of these genes which have previously been linked to immune and inflammation-related pathways have not been reported in neuropathic pain (e.g., Laptm5, Fcgr3a). CONCLUSIONS: Our results revealed that immune and defense pathways may contribute to the generation of neuropathic pain after CCI. These mRNAs may represent new therapeutic targets for the treatment of neuropathic pain.