Identification of differentially expressed mRNA/lncRNA modules in acutely regorafenib-treated sorafenib-resistant Huh7 hepatocellular carcinoma cells.

The multikinase inhibitor sorafenib is the standard first-line treatment for advanced hepatocellular carcinoma (HCC), but many patients become sorafenib-resistant (SR). This study investigated the efficacy of another kinase inhibitor, regorafenib (Rego), as a second-line treatment. We produced SR HCC cells, wherein the PI3K-Akt, TNF, cAMP, and TGF-beta signaling pathways were affected. Acute Rego treatment of these cells reversed the expression of genes involved in TGF-beta signaling but further increased the expression of genes involved in PI3K-Akt signaling. Additionally, Rego reversed the expression of genes involved in nucleosome assembly and epigenetic gene expression. Weighted gene co-expression network analysis (WGCNA) revealed four differentially expressed long non-coding RNA (DElncRNA) modules that were associated with the effectiveness of Rego on SR cells. Eleven putative DElncRNAs with distinct expression patterns were identified. We associated each module with DEmRNAs of the same pattern, thus obtaining DElncRNA/DEmRNA co-expression modules. We discuss the potential significance of each module. These findings provide insights and resources for further investigation into the potential mechanisms underlying the response of SR HCC cells to Rego.

Simultaneous determination of L-tryptophan impurities in meat products.

L-tryptophan has been used as a feed additive for swine and poultry and as a nutrient supplement for humans. However, some impurities in L-tryptophan have been reported as causative components of eosinophilia-myalgia syndrome. Therefore, from a safety perspective, it is important to analyze meat samples for these impurities. This study aims to develop an analytical method for the simultaneous detection of L-tryptophan impurities in meat products using LC-MS/MS. Among the various impurities, detection methods for (S)-2-amino-3-(5-hydroxy-1H-indol-3-yl)propanoic acid (5-hydroxytryptophan) (HTP), 1-methyl-1,2,3,4-tetrahydro-ß-carboline-3-carboxylic acid (MTCA), 3a-hydroxy-1,2,3,3a,8,8a-hexahydropyrrolo-[2,3-b]-indole-2-carboxylic acid (PIC), and 1,1'-ethylidenebistryptophan (EBT) and 2-(3-indoylmethyl)-L-tryptophan (IMT) were developed. The developed method allowed simultaneous determination of these four impurities in 5 min. No interferences from the matrix were observed, and the method showed good sensitivity to each analyte. The method detection limit and limit of quantification in meat matrices were below 11.2 and 35.7 µg/kg, respectively.

Analysis of differentially expressed long non-coding RNAs in LPS-induced human HMC3 microglial cells.

BACKGROUND: Long non-coding RNAs (lncRNAs) are emerging as key modulators of inflammatory gene expression, but their roles in neuroinflammation are poorly understood. Here, we identified the inflammation-related lncRNAs and correlated mRNAs of the lipopolysaccharide (LPS)-treated human microglial cell line HMC3. We explored their potential roles and interactions using bioinformatics tools such as gene ontology (GO), kyoto encyclopedia of genes and genomes (KEGG), and weighted gene co-expression network analysis (WGCNA). RESULTS: We identified 5 differentially expressed (DE) lncRNAs, 4 of which (AC083837.1, IRF1-AS1, LINC02605, and MIR3142HG) are novel for microglia. The DElncRNAs with their correlated DEmRNAs (99 total) fell into two network modules that both were enriched with inflammation-related RNAs. However, treatment with the anti-inflammatory agent JQ1, an inhibitor of the bromodomain and extra-terminal (BET) protein BRD4, neutralized the LPS effect in only one module, showing little or even enhancing effect on the other. CONCLUSIONS: These results provide insight into, and a resource for studying, the regulation of microglia-mediated neuroinflammation and its potential therapy by small-molecule BET inhibitors.

The bromodomain inhibitor JQ1 up-regulates the long non-coding RNA MALAT1 in cultured human hepatic carcinoma cells.

The epigenetic reader, bromodomain-containing 4 (BRD4), is overexpressed in hepatocellular carcinoma (HCC), and BRD4 inhibition is considered as a new therapeutic approach. The BRD inhibitor JQ1 is known to inhibit the enrichment of BRD4 at enhancer sites. Gene network analyses have implicated long non-coding RNAs (lncRNAs) in the effects of JQ1, but the precise molecular events remain unexplored. Here, we report that in HepG2 cells, JQ1 significantly reduced various proliferation-related lncRNAs, but up-regulated the known liver tumor marker, MALAT1. Using ChIP-sequencing data, ChIP-qPCR, luciferase reporter assays, and chromatin conformation capture (3C), we characterized the MALAT1 gene locus. We found that JQ1 elicited a rearrangement of its chromatin looping conformation, which involved the putative enhancers E1, E2, E3, the gene body, and the promoter. We further found that the forkhead box protein A2 (FOXA2) binds to E2 and the promoter; suppression of FOXA2 expression resulted in MALAT1 up-regulation and increased cell proliferation. These results suggest that the inhibition of MALAT1 may improve the effect of BET inhibitors as an anti-cancer therapy and that FOXA2 would be a suitable target for that approach.

Comprehensive transcriptome profiling of BET inhibitor-treated HepG2 cells.

Hepatocellular carcinoma (HCC) is the most common primary liver cancer and poor prognosis. Emerging evidence suggests that epigenetic alterations play a crucial role in HCC, suggesting epigenetic inhibition as a promising therapeutic approach. Indeed, the bromodomain and extra-terminal (BET) inhibitors inhibit the proliferation and invasion of various cancers but still lack a strong mechanistic rationale. Here, we identified the differentially expressed mRNAs (DEmRNAs) and lncRNAs (DElncRNAs) in human HCC cell line HepG2 treated with the BET inhibitors, JQ1, OTX015, or ABBV-075. We analyzed the correlation between DEmRNAs and DElncRNAs in common for the three inhibitors based on their expression profiles and performed functional annotation pathway enrichment analysis. Most of these shared DEmRNAs and DElncRNAs, including some novel transcripts, were downregulated, indicating decreased proliferation/adhesion and increased apoptosis/inflammation. Our study suggests that BET proteins play a crucial role in regulating cancer progression-related genes and provide a valuable resource for novel putative biomarkers and therapeutic targets in HCC.

Targeting of noncoding RNAs encoded by a novel MYC enhancers inhibits the proliferation of human hepatic carcinoma cells in vitro.

The proto-oncogene MYC is important for development and cell growth, however, its abnormal regulation causes cancer. Recent studies identified distinct enhancers of MYC in various cancers, but any MYC enhancer(s) in hepatocellular carcinoma (HCC) remain(s) elusive. By analyzing H3K27ac enrichment and enhancer RNA (eRNA) expression in cultured HCC cells, we identified six putative MYC enhancer regions. Amongst these, two highly active enhancers, located ~ 800 kb downstream of the MYC gene, were identified by qRT-PCR and reporter assays. We functionally confirmed these enhancers by demonstrating a significantly reduced MYC expression and cell proliferation upon CRISPR/Cas9-based deletion and/or antisense oligonucleotide (ASO)-mediated inhibition. In conclusion, we identified potential MYC enhancers of HCC and propose that the associated eRNAs may be suitable targets for HCC treatment.

BET inhibitor suppresses migration of human hepatocellular carcinoma by inhibiting SMARCA4.

Hepatocellular carcinoma (HCC) is one of the most prevalent and poorly responsive cancers worldwide. Bromodomain and extraterminal (BET) inhibitors, such as JQ1 and OTX-015, inhibit BET protein binding to acetylated residues in histones. However, the physiological mechanisms and regulatory processes of BET inhibition in HCC remain unclear. To explore BET inhibitors' potential role in the molecular mechanisms underlying their anticancer effects in HCC, we analyzed BET inhibitor-treated HCC cells' gene expression profiles with RNA-seq and bioinformatics analysis. BET inhibitor treatment significantly downregulated genes related to bromodomain-containing proteins 4 (BRD4), such as ACSL5, SLC38A5, and ICAM2. Importantly, some cell migration-related genes, including AOC3, CCR6, SSTR5, and SCL7A11, were significantly downregulated. Additionally, bioinformatics analysis using Ingenuity Knowledge Base Ingenuity Pathway Analysis (IPA) revealed that SMARCA4 regulated migration response molecules. Furthermore, knockdown of SMARCA4 gene expression by siRNA treatment significantly reduced cell migration and the expression of migration-related genes. In summary, our results indicated that BET inhibitor treatment in HCC cell lines reduces cell migration through the downregulation of SMARCA4.

The BET inhibitor attenuates the inflammatory response and cell migration in human microglial HMC3 cell line.

Microglia, resident macrophages of the brain that act as primary immune cells, play essential roles in innate immunity and neuroinflammatory pathologies. Microglial cells are rapidly activated in response to infection and inflammation/injury, associated with the expression of proinflammatory genes and secretion of cytokines. The bromodomain and extra-terminal (BET) inhibitor JQ1 has been shown to be an epigenetic agent that reduces inflammation. In this study, we investigated the mechanisms underlying the anti-inflammatory and anti-migratory functions of JQ1 and the genes targeted by JQ1 in lipopolysaccharide (LPS)-activated human microglial clone 3 (HMC3) cells using RNA-sequencing (RNA-seq). We analyzed the pattern of inflammation-related genes (chemokines, cytokines, and interferon-stimulated genes) and migration-related genes with JQ1 treatment from differentially expressed genes analysis in HMC3 cells. We found that LPS-induced IRF1 directly regulated inflammation- and migration-related genes and that JQ1 significantly reduced IRF1 and its target genes. Additionally, IRF1 attenuation significantly downregulated target genes and inhibited microglial migration. Our data suggest that the BET inhibitor JQ1 can modulate the inflammatory response and migration through the regulation of LPS-induced IRF1 in human microglia.

Inhibition of the DevSR Two-Component System by Overexpression of Mycobacterium tuberculosis PknB in Mycobacterium smegmatis.

The DevSR (DosSR) two-component system, which is a major regulatory system involved in oxygen sensing in mycobacteria, plays an important role in hypoxic induction of many genes in mycobacteria. We demonstrated that overexpression of the kinase domain of Mycobacterium tuberculosis (Mtb) PknB inhibited transcriptional activity of the DevR response regulator in Mycobacterium smegmatis and that this inhibitory effect was exerted through phosphorylation of DevR on Thr180 within its DNA-binding domain. Moreover, the purified kinase domain of Mtb PknB significantly phosphorylated RegX3, NarL, KdpE, TrcR, DosR, and MtrA response regulators of Mtb that contain the Thr residues corresponding to Thr180 of DevR in their DNA-binding domains, implying that transcriptional activities of these response regulators might also be inhibited when the kinase domain of PknB is overexpressed.

Development of a ubiquitous healthcare monitoring system combined with non-conscious and ambulatory physiological measurements and its application to medical care.

The demand for ubiquitous healthcare monitoring has been increasingly raised for prevention of lifestyle-related diseases, acute life support or chronic therapies for inpatients and/or outpatients having chronic disorder and home medical care. From these view points, we developed a non-conscious healthcare monitoring system without any attachment of biological sensors and operations of devices, and an ambulatory postural changes and activities monitoring system. Furthermore in this study, in order to investigate those applicability to the ubiquitous healthcare monitoring, we have developed a new healthcare monitoring system combined with the non-conscious and the ambulatory measurements developed by us. In patients with chronic cardiovascular disease or stroke, the daily health conditions such as pulse, respiration, activities and so on, could be continuously measured in the hospital, the rehabilitation room and subject's own home, using the present system. The results demonstrated that the system appears useful for the ubiquitous healthcare monitoring not only at medical facility, but also during daily living at home.