Overexpression of heat shock protein 47 is associated with increased proliferation and metastasis in gastric cancer.

Here, we investigated that the heat shock protein 47 (HSP47) plays a crucial role in the progression of gastric cancer (GC). We analyzed HSP47 gene expression in GC cell lines and patient tissues. The HSP47 mRNA and protein expression levels were significantly higher in GC cell lines and tumor tissues compared to normal gastric mucosa. Using siRNA to silence the expression of HSP47 in GC cells resulted in a significant reduction in their proliferation, wound healing, migration, and invasion capacities. Additionally, we also showed that the mRNA expression of matrix metallopeptidase-7 (MMP-7), a metastasis-promoting gene, was significantly reduced in HSP47 siRNA-transfected GC cells. We confirmed that the HSP47 promoter region was methylated in the SNU-216 GC cell line expressing low levels of HSP47 and in most non-cancerous gastric tissues. It means that the expression of HSP47 is regulated by epigenetic regulatory mechanisms. These findings suggest that targeting HSP47, potentially through its promoter methylation, could be a useful new therapeutic strategy for treating GC.

The preventive effect of Gastrodia elata Blume extract on vancomycin-induced acute kidney injury in rats.

BACKGROUND: Gastrodia elata Blume (GEB), a traditional medicinal herb, has been reported to have pharmacological effect including protection against liver, neuron and kidney toxicity. However, explanation of its underlying mechanisms remains a great challenge. This study investigated the protective effects of GEB extract on vancomycin (VAN)-induced nephrotoxicity in rats and underlying mechanisms with emphasis on the anti-oxidative stress, anti-inflammation and anti-apoptosis. The male Sprague-Dawley rats were randomly divided three groups: control (CON) group, VAN group and GEB group with duration of 14 days. RESULTS: The kidney weight and the serum levels of blood urea nitrogen and creatinine in the GEB group were lower than the VAN group. Histological analysis using hematoxylin & eosin and periodic acid Schiff staining revealed pathological changes of the VAN group. Immunohistochemical analysis revealed that the expression levels of N-acetyl-D-glucosaminidase, myeloperoxidase and tumor necrosis factor-alpha in the GEB group were decreased when compared with the VAN group. The number of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling-positive cells, phosphohistone and malondialdehyde levels were lower in the GEB group than VAN group. The levels of total glutathione in the GEB group were higher than the VAN group. CONCLUSIONS: The findings of this study suggested that GEB extract prevents VAN-induced renal tissue damage through anti-oxidation, anti-inflammation and anti-apoptosis.

Integrative transcriptomic and genomic analyses unveil the IFI16 variants and expression as MASLD progression markers.

BACKGROUND AND AIMS: Metabolic dysfunction-associated steatotic liver disease (MASLD) encompasses a broad and continuous spectrum of liver diseases ranging from fatty liver to steatohepatitis. The intricate interactions of genetic, epigenetic, and environmental factors in the development and progression of MASLD remain elusive. Here, we aimed to achieve an integrative understanding of the genomic and transcriptomic alterations throughout the progression of MASLD. APPROACH AND RESULTS: RNA-Seq profiling (n = 146) and whole-exome sequencing (n = 132) of MASLD liver tissue samples identified 3 transcriptomic subtypes (G1-G3) of MASLD, which were characterized by stepwise pathological and molecular progression of the disease. Macrophage-driven inflammatory activities were identified as a key feature for differentiating these subtypes. This subtype-discriminating macrophage interplay was significantly associated with both the expression and genetic variation of the dsDNA sensor IFI16 (rs6940, A>T, T779S), establishing it as a fundamental molecular factor in MASLD progression. The in vitro dsDNA-IFI16 binding experiments and structural modeling revealed that the IFI16 variant exhibited increased stability and stronger dsDNA binding affinity compared to the wild-type. Further downstream investigation suggested that the IFI16 variant exacerbated DNA sensing-mediated inflammatory signals through mitochondrial dysfunction-related signaling of the IFI16-PYCARD-CASP1 pathway. CONCLUSIONS: This study unveils a comprehensive understanding of MASLD progression through transcriptomic classification, highlighting the crucial roles of IFI16 variants. Targeting the IFI16-PYCARD-CASP1 pathway may pave the way for the development of novel diagnostics and therapeutics for MASLD.

Identification of signature gene set as highly accurate determination of metabolic dysfunction-associated steatotic liver disease progression.

BACKGROUND/AIMS: Metabolic dysfunction-associated steatotic liver disease (MASLD) is characterized by fat accumulation in the liver. MASLD encompasses both steatosis and MASH. Since MASH can lead to cirrhosis and liver cancer, steatosis and MASH must be distinguished during patient treatment. Here, we investigate the genomes, epigenomes, and transcriptomes of MASLD patients to identify signature gene set for more accurate tracking of MASLD progression. METHODS: Biopsy-tissue and blood samples from patients with 134 MASLD, comprising 60 steatosis and 74 MASH patients were performed omics analysis. SVM learning algorithm were used to calculate most predictive features. Linear regression was applied to find signature gene set that distinguish the stage of MASLD and to validate their application into independent cohort of MASLD. RESULTS: After performing WGS, WES, WGBS, and total RNA-seq on 134 biopsy samples from confirmed MASLD patients, we provided 1,955 MASLD-associated features, out of 3,176 somatic variant callings, 58 DMRs, and 1,393 DEGs that track MASLD progression. Then, we used a SVM learning algorithm to analyze the data and select the most predictive features. Using linear regression, we identified a signature gene set capable of differentiating the various stages of MASLD and verified it in different independent cohorts of MASLD and a liver cancer cohort. CONCLUSION: We identified a signature gene set (i.e., CAPG, HYAL3, WIPI1, TREM2, SPP1, and RNASE6) with strong potential as a panel of diagnostic genes of MASLD-associated disease.

SARS-CoV-2 spike protein accelerates systemic sclerosis by increasing inflammatory cytokines, Th17 cells, and fibrosis.

BACKGROUND: Coronavirus disease 2019 (COVID-19) induces a dysfunctional immune response, inflammation, autoantibody production, and coagulopathy, which are symptoms that bear resemblance to those of autoimmune diseases, including systemic sclerosis (SSc). METHODS: While there is a single case report suggesting an association between COVID-19 and SSc, the effects of COVID-19 on SSc are not yet fully understood. Human embryonic kidney 293 (HEK293) cells were transfected with the SARS-CoV-2 spike protein gene, in the presence of TGF-ß. The expression levels of fibrosis-related proteins were measured via Western blotting. A bleomycin (BLM)-induced SSc mouse model was employed, wherein mice were injected with the gene encoding the SARS-CoV-2 spike protein and the ACE2 receptor. The levels of fibrosis, autoantibodies, thrombotic factors, and inflammatory cytokines in tissues and serum were analyzed. RESULTS: In vitro, the expression levels of fibrosis marker proteins were elevated in the spike protein group compared to the control group. In vivo, the skin thickness of SSc mice increased following exposure to the SARS-CoV-2 spike protein. Furthermore, the levels of autoantibodies and thrombotic factors, such as anti-phospholipid antibodies (APLA), were significantly increased in the presence of the protein. Flow cytometry analysis revealed increased expression of the proinflammatory cytokine IL-17 in the skin, lungs, and blood. Moreover, tissue fibrosis and levels of inflammatory cytokines in skin and lung tissues were markedly escalated in SSc mice subjected to the protein. CONCLUSION: COVID-19 may accelerate the development and progression of SSc by intensifying fibrosis through the upregulation of inflammation, autoantibody production, and thrombosis.

Lactobacillus acidophilus and propionate attenuate Sjögren's syndrome by modulating the STIM1-STING signaling pathway.

BACKGROUND: Sjögren's syndrome (SS) is an autoimmune disease characterized by inflammation of the exocrine gland. An imbalance of gut microbiota has been linked to SS. However, the molecular mechanism is unclear. We investigated the effects of Lactobacillus acidophilus (L. acidophilus) and propionate on the development and progression of SS in mouse model. METHODS: We compared the gut microbiomes of young and old mice. We administered L. acidophilus and propionate up to 24 weeks. The saliva flow rate and the histopathology of the salivary glands were investigated, and the effects of propionate on the STIM1-STING signaling pathway were evaluated in vitro. RESULTS: Lactobacillaceae and Lactobacillus were decreased in aged mice. SS symptoms were ameliorated by L. acidophilus. The abundance of propionate-producing bacterial was increased by L. acidophilus. Propionate ameliorated the development and progression of SS by inhibiting the STIM1-STING signaling pathway. CONCLUSIONS: The findings suggest that Lactobacillus acidophilus and propionate have therapeutic potential for SS. Video Abstract.

nc886, an RNA Polymerase III-Transcribed Noncoding RNA Whose Expression Is Dynamic and Regulated by Intriguing Mechanisms.

nc886 is a medium-sized non-coding RNA that is transcribed by RNA polymerase III (Pol III) and plays diverse roles in tumorigenesis, innate immunity, and other cellular processes. Although Pol III-transcribed ncRNAs were previously thought to be expressed constitutively, this concept is evolving, and nc886 is the most notable example. The transcription of nc886 in a cell, as well as in human individuals, is controlled by multiple mechanisms, including its promoter CpG DNA methylation and transcription factor activity. Additionally, the RNA instability of nc886 contributes to its highly variable steady-state expression levels in a given situation. This comprehensive review discusses nc886's variable expression in physiological and pathological conditions and critically examines the regulatory factors that determine its expression levels.

SARS-CoV-2 spike protein promotes inflammatory cytokine activation and aggravates rheumatoid arthritis.

BACKGROUND: Coronavirus disease 2019 (COVID-19) induces inflammation, autoantibody production, and thrombosis, which are common symptoms of autoimmune diseases, including rheumatoid arthritis (RA). However, the effect of COVID-19 on autoimmune disease is not yet fully understood. METHODS: This study was performed to investigate the effects of COVID-19 on the development and progression of RA using a collagen-induced arthritis (CIA) animal model. Human fibroblast-like synoviocytes (FLS) were transduced with lentivirus carrying the SARS-CoV-2 spike protein gene in vitro, and the levels of inflammatory cytokine and chemokine expression were measured. For in vivo experiments, CIA mice were injected with the gene encoding SARS-CoV-2 spike protein, and disease severity, levels of autoantibodies, thrombotic factors, and inflammatory cytokine and chemokine expression were assessed. In the in vitro experiments, the levels of inflammatory cytokine and chemokine expression were significantly increased by overexpression of SARS-CoV-2 spike protein in human FLS. RESULTS: The incidence and severity of RA in CIA mice were slightly increased by SARS-CoV-2 spike protein in vivo. In addition, the levels of autoantibodies and thrombotic factors, such as anti-CXC chemokine ligand 4 (CXCL4, also called PF4) antibodies and anti-phospholipid antibodies were significantly increased by SARS-CoV-2 spike protein. Furthermore, tissue destruction and inflammatory cytokine level in joint tissue were markedly increased in CIA mice by SARS-CoV-2 spike protein. CONCLUSIONS: The results of the present study suggested that COVID-19 accelerates the development and progression of RA by increasing inflammation, autoantibody production, and thrombosis. Video Abstract.

Mitochondrial Transplantation Ameliorates the Development and Progression of Osteoarthritis.

Osteoarthritis (OA) is a common degenerative joint disease characterized by breakdown of joint cartilage. Mitochondrial dysfunction of the chondrocyte is a risk factor for OA progression. We examined the therapeutic potential of mitochondrial transplantation for OA. Mitochondria were injected into the knee joint of monosodium iodoacetate-induced OA rats. Chondrocytes from OA rats or patients with OA were cultured to examine mitochondrial function in cellular pathophysiology. Pain, cartilage destruction, and bone loss were improved in mitochondrial transplanted-OA rats. The transcript levels of IL-1ß, TNF-α, matrix metallopeptidase 13, and MCP-1 in cartilage were markedly decreased by mitochondrial transplantation. Mitochondrial function, as indicated by membrane potential and oxygen consumption rate, in chondrocytes from OA rats was improved by mitochondrial transplantation. Likewise, the mitochondrial function of chondrocytes from OA patients was improved by coculture with mitochondria. Furthermore, inflammatory cell death was significantly decreased by coculture with mitochondria. Mitochondrial transplantation ameliorated OA progression, which is caused by mitochondrial dysfunction. These results suggest the therapeutic potential of mitochondrial transplantation for OA.

A host non-coding RNA, nc886, plays a pro-viral role by promoting virus trafficking to the nucleus.

Elucidation of the interplay between viruses and host cells is crucial for taming viruses to benefit human health. Cancer therapy using adenovirus, called oncolytic virotherapy, is a promising treatment option but is not robust in all patients. In addition, inefficient replication of human adenovirus in mouse hampered the development of an in vivo model for preclinical evaluation of therapeutically engineered adenovirus. nc886 is a human non-coding RNA that suppresses Protein Kinase R (PKR), an antiviral protein. In this study, we have found that nc886 greatly promotes adenoviral gene expression and replication. Remarkably, the stimulatory effect of nc886 is not dependent on its function to inhibit PKR. Rather, nc886 facilitates the nuclear entry of adenovirus via modulating the kinesin pathway. nc886 is not conserved in mouse and, when xenogeneically expressed in mouse cells, promotes adenovirus replication. Our investigation has discovered a novel mechanism of how a host ncRNA plays a pro-adenoviral role. Given that nc886 expression is silenced in a subset of cancer cells, our study highlights that oncolytic virotherapy might be inefficient in those cells. Furthermore, our findings open future possibilities of harnessing nc886 to improve the efficacy of oncolytic adenovirus and to construct nc886-expressing transgenic mice as an animal model.

Nc886, a Novel Suppressor of the Type I Interferon Response Upon Pathogen Intrusion.

Interferons (IFNs) are a crucial component in the innate immune response. Especially the IFN-ß signaling operates in most cell types and plays a key role in the first line of defense upon pathogen intrusion. The induction of IFN-ß should be tightly controlled, because its hyperactivation can lead to tissue damage or autoimmune diseases. Activation of the IFN-ß promoter needs Interferon Regulatory Factor 3 (IRF3), together with Nuclear Factor kappa-light-chain-enhancer of activated B cells (NF-κB) and Activator Protein 1 (AP-1). Here we report that a human noncoding RNA, nc886, is a novel suppressor for the IFN-ß signaling and inflammation. Upon treatment with several pathogen-associated molecular patterns and viruses, nc886 suppresses the activation of IRF3 and also inhibits NF-κB and AP-1 via inhibiting Protein Kinase R (PKR). These events lead to decreased expression of IFN-ß and resultantly IFN-stimulated genes. nc886's role might be to restrict the IFN-ß signaling from hyperactivation. Since nc886 expression is regulated by epigenetic and environmental factors, nc886 might explain why innate immune responses to pathogens are variable depending on biological settings.

A Regulatory Noncoding RNA, nc886, Suppresses Esophageal Cancer by Inhibiting the AKT Pathway and Cell Cycle Progression.

nc886 is a regulatory non-coding RNA (ncRNA) whose expression is frequently silenced in malignancies. In the case of esophageal squamous cell carcinoma (ESCC), nc886 silencing is associated with shorter survival of patients, suggesting nc886's tumor suppressor role in ESCC. However, this observation has not been complemented by an in-detail study about nc886's impact on gene expression and cellular phenotypes. Here we have shown that nc886 inhibits AKT, a key protein in a renowned pro-survival pathway in cancer. nc886-silenced cells (nc886- cells) have activated AKT and altered expression of cell cycle genes. nc886- cells tend to have lower expression of CDKN2A and CDKN2C, both of which are inhibitors for cyclin-dependent kinase (CDK), and higher expression of CDK4 than nc886-expressing cells. As a result, nc886- cells are hyperactive in the progression of the G1 to S cell cycle phase, proliferate faster, and are more sensitive to palbociclib, which is a cancer therapeutic drug that targets CDK4/6. Experimentally by nc886 expression and knockdown, we have determined the AKT target genes and cell cycle genes that are controlled by nc886 (nc886-associated gene sets). These gene sets, in combination with pathologic staging and nc886 expression levels, are a vastly superior predictor for the survival of 108 ESCC patients. In summary, our study has elucidated in ESCC how nc886 inhibits cell proliferation to explain its tumor suppressor role and identified gene sets that are of future clinical utility, by predicting patient survival and responsiveness to a therapeutic drug.

Protein kinase R and its cellular regulators in cancer: An active player or a surveillant?

Protein kinase R (PKR), originally known as an antiviral protein, senses various stresses as well as pathogen-driven double-stranded RNAs. Thereby activated PKR provokes diverse downstream events, including eIF2α phosphorylation and nuclear factor kappa-light-chain-enhancer of activated B cells activation. Consequently, PKR induces apoptosis and inflammation, both of which are highly important in cancer as much as its original antiviral role. Therefore, cellular proteins and RNAs should tightly control PKR activity. PKR and its regulators are often dysregulated in cancer and it is undoubted that such dysregulation contributes to tumorigenesis. However, PKR's precise role in cancer is still in debate, due to incomprehensible and even contradictory data. In this review, we introduce important cellular PKR regulators and discuss about their roles in cancer. Among them, we pay particular attention to nc886, a PKR repressor noncoding RNA that has been identified relatively recently, because its expression pattern in cancer can explain interesting yet obscure oncologic aspects of PKR. Based on nc886 and its regulation of PKR, we have proposed a tumor surveillance model, which reconciles contradictory data about PKR in cancer. This article is categorized under: Regulatory RNAs/RNAi/Riboswitches > Regulatory RNAs RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications.

Mechanism mediated by a noncoding RNA, nc886, in the cytotoxicity of a DNA-reactive compound.

DNA-reactive compounds are harnessed for cancer chemotherapy. Their genotoxic effects are considered to be the main mechanism for the cytotoxicity to date. Because this mechanism preferentially affects actively proliferating cells, it is postulated that the cytotoxicity is specific to cancer cells. Nonetheless, they do harm normal quiescent cells, suggesting that there are other cytotoxic mechanisms to be uncovered. By employing doxorubicin as a representative DNA-reactive compound, we have discovered a cytotoxic mechanism that involves a cellular noncoding RNA (ncRNA) nc886 and protein kinase R (PKR) that is a proapoptotic protein. nc886 is transcribed by RNA polymerase III (Pol III), binds to PKR, and prevents it from aberrant activation in most normal cells. We have shown here that doxorubicin evicts Pol III from DNA and, thereby, shuts down nc886 transcription. Consequently, the instantaneous depletion of nc886 provokes PKR and leads to apoptosis. In a short-pulse treatment of doxorubicin, these events are the main cause of cytotoxicity preceding the DNA damage response in a 3D culture system as well as the monolayer cultures. By identifying nc886 as a molecular signal for PKR to sense doxorubicin, we have provided an explanation for the conundrum why DNA-damaging drugs can be cytotoxic to quiescent cells that have the competent nc886/PKR pathway.

Author Correction: nc886 is induced by TGF-ß and suppresses the microRNA pathway in ovarian cancer.

In the original version of the Supplementary Information file associated with this Article, Supplementary Fig. 18 panel b was inadvertently replaced with a duplicate of panel a. The error has now been fixed and the corrected version of the Supplementary Information PDF is available to download from the HTML version of the Article.

nc886 is induced by TGF-ß and suppresses the microRNA pathway in ovarian cancer.

Transforming growth factor-ß (TGF-ß) signaling and microRNAs (miRNAs) are important gene regulatory components in cancer. Usually in advanced malignant stages, TGF-ß signaling is elevated but global miRNA expression is suppressed. Such a gene expression signature is well illustrated in a fibrosis (or mesenchymal) subtype of ovarian cancer (OC) that is of poor prognosis. However, the interplay between the two pathways in the OC subtype has not yet been elucidated. nc886 is a recently identified non-coding RNA implicated in several malignancies. The high expression of nc886 is associated with poor prognosis in 285 OC patients. Herein, we find that in OC nc886 expression is induced by TGF-ß and that nc886 binds to Dicer to inhibit miRNA maturation. By preventing the miRNA pathway, nc886 emulates TGF-ß in gene expression patterns and potentiates cell adhesion, migration, invasion, and drug resistance. Here we report nc886 to be a molecular link between the TGF-ß and miRNA pathways.

Epigenetic regulation of noncoding RNA transcription by mammalian RNA polymerase III.

RNA polymerase III (Pol III) synthesizes a range of medium-sized noncoding RNAs (collectively 'Pol III genes') whose early established biological roles were so essential that they were considered 'housekeeping genes'. Besides these fundamental functions, diverse unconventional roles of mammalian Pol III genes have recently been recognized and their expression must be exquisitely controlled. In this review, we summarize the epigenetic regulation of Pol III genes by chromatin structure, histone modification and CpG DNA methylation. We also recapitulate the association between dysregulation of Pol III genes and diseases such as cancer and neurological disorders. Additionally, we will discuss why in-depth molecular studies of Pol III genes have not been attempted and how nc886, a Pol III gene, may resolve this issue.

Oncogenic function and clinical implications of SLC3A2-NRG1 fusion in invasive mucinous adenocarcinoma of the lung.

The neuregulin 1 (NRG1) fusion is a recently identified novel driver oncogene in invasive mucinous adenocarcinoma of the lung (IMA). After identification of a case of SLC3A2-NRG1 in a patient with IMA, we verified this fusion gene in a cohort of 59 patients with IMA. Targeted cancer panel sequencing and RT-PCR identified the possible coexistence of other driver oncogenes. Among 59 IMAs, we found 16 NRG1 fusions (13 SLC3A2-NRG1 and 3 CD74-NRG1). Of 16 patients with NRG1 fusions, concurrent KRAS codon 12 mutations were found in 10 cases. We also found concurrent NRAS Q61L mutation and EML4-ALK fusion in additional two cases with NRG1 fusions. When comparing overall survival (OS) according to the presence of NRG1 fusions showed that patients harboring NRG1 fusions had significantly inferior OS than those without NRG1 fusions (hazard ratio = 0.286; 95% confidence interval, .094 to .865). Ectopic expression of the SLC3A2-NRG1 fusion in lung cancer cells increased cell migration, proliferation and tumor growth in vitro and in xenograft models, suggesting oncogenic function for the fusion protein. We found that the SLC3A2-NRG1 fusion promoted ERBB2-ERBB3 phosphorylation and heteroduplex formation and activated the downstream PI3K/AKT/mTOR pathway through paracrine signaling. These findings suggested that the SLC3A2-NRG1 fusion was a driver in IMA with an important prognostic impact. SLC3A2-NRG1 should be considered a therapeutic target for patients with IMA.

nc886, a non-coding RNA and suppressor of PKR, exerts an oncogenic function in thyroid cancer.

nc886 is a recently identified cellular non-coding RNA and its depletion leads to acute cell death via PKR (Protein Kinase RNA-activated) activation. nc886 expression is increased in some malignancies, but silenced in others. However, the precise role of nc886/PKR is controversial: is it a tumor suppressor or an oncogene? In this study, we have clarified the role of nc886 in thyroid cancer by sequentially generating PKR knockout (KO) and PKR/nc886 double KO cell lines from Nthy-ori 3-1, a partially transformed thyroid cell line. Compared to the wildtype, PKR KO alone does not exhibit any significant phenotypic changes. However, nc886 KO cells are less proliferative, migratory, and invasive than their parental PKR KO cells. Importantly, the requirement of nc886 in tumor phenotypes is totally independent of PKR. In our microarray data, nc886 KO suppresses some genes whose elevated expression is associated with poor survival confirmed by data from total of 505 thyroid cancer patients in the Caner Genome Atlas project. Also, the nc886 expression level tends to be elevated and in more aggressively metastatic tumor specimens from thyroid cancer patients. In summary, we have discovered nc886's tumor-promoting role in thyroid cancer which has been concealed by the PKR-mediated acute cell death.