Negative regulation of SH2B3 by SMYD5 controls epithelial-mesenchymal transition in lung cancer.

The main cause of death in lung cancer patients is metastasis. Thus, efforts to suppress micrometastasis or distant metastasis in lung cancer, identify therapeutic targets and develop related drugs are ongoing. In this study, we identified SMYD5 as a novel metastasis regulator in lung cancer and found that SMYD5 was overexpressed in lung cancer based on both RNA-sequencing (RNA-seq) analysis results derived from the TCGA portal and immunohistochemical analysis results; knockdown of SMYD5 inhibited cell migration and invasion by changing EMT (epithelial-mesenchymal transition) markers and MMP9 expression in NCI-H1299 and H1703 cell lines. Additionally, SMYD5 knockdown increased SH2B3 expression by decreasing the level of H4K20 trimethylation. Furthermore, in an in vitro EMT system using TGF-ß treatment, SMYD5 knockdown resulted in reduced cell migration and invasion in the highly invasive NCI-H1299 and H1703 cell lines. Based on these findings, we propose that SMYD5 could serve as a potential therapeutic target for lung cancer treatment and that cotreatment with an SMYD5 inhibitor and chemotherapy may enhance the therapeutic effect of lung cancer treatment.

Chemically-defined and scalable culture system for intestinal stem cells derived from human intestinal organoids.

Three-dimensional human intestinal organoids (hIO) are widely used as a platform for biological and biomedical research. However, reproducibility and challenges for large-scale expansion limit their applicability. Here, we establish a human intestinal stem cell (ISC) culture method expanded under feeder-free and fully defined conditions through selective enrichment of ISC populations (ISC3D-hIO) within hIO derived from human pluripotent stem cells. The intrinsic self-organisation property of ISC3D-hIO, combined with air-liquid interface culture in a minimally defined medium, forces ISC3D-hIO to differentiate into the intestinal epithelium with cellular diversity, villus-like structure, and barrier integrity. Notably, ISC3D-hIO is an ideal cell source for gene editing to study ISC biology and transplantation for intestinal diseases. We demonstrate the intestinal epithelium differentiated from ISC3D-hIO as a model system to study severe acute respiratory syndrome coronavirus 2 viral infection. ISC3D-hIO culture technology provides a biological tool for use in regenerative medicine and disease modelling.

Efficient and reproducible generation of human induced pluripotent stem cell-derived expandable liver organoids for disease modeling.

Genetic liver disease modeling is difficult because it is challenging to access patient tissue samples and to develop practical and relevant model systems. Previously, we developed novel proliferative and functional liver organoids from pluripotent stem cells; however, the protocol requires improvement for standardization and reproducible mass production. Here, we improved the method such that it is suitable for scalable expansion and relatively homogenous production, resulting in an efficient and reproducible process. Moreover, three medium components critical for long-term expansion were defined. Detailed transcriptome analysis revealed that fibroblast growth factor signaling, the essential pathway for hepatocyte proliferation during liver regeneration, was mainly enriched in proliferative liver organoids. Short hairpin RNA-mediated knockdown of FGFR4 impaired the generation and proliferation of organoids. Finally, glycogen storage disease type Ia (GSD1a) patient-specific liver organoids were efficiently and reproducibly generated using the new protocol. They well maintained disease-specific phenotypes such as higher lipid and glycogen accumulation in the liver organoids and lactate secretion into the medium consistent with the main pathologic characteristics of patients with GSD1a. Therefore, our newly established liver organoid platform can provide scalable and practical personalized disease models and help to find new therapies for incurable liver diseases including genetic liver diseases.

The bone-protective benefits of amino-conjugated calcium in an ovariectomized (OVX) rat model.

Low bone density, fragility, and microarchitectural disintegration are the symptoms of osteoporosis. An imbalance between bone growth and resorption can lead to osteoporosis. This study evaluated the effects of amino-calcium (AC) on bone protection in ovariectomized control group (NC) rats. Amino-calcium (AC) was characterized using Fourier-transform infrared spectroscopy (FT-IR), energy-dispersive X-ray spectroscopy (EDS), and nuclear magnetic resonance spectroscopy analyses (NMR). After determining the biocompatibility of amino-calcium (AC) with MC3T3-E1 cells, alkaline phosphatase staining revealed significant changes on day 7. Three of the four groups underwent ovariectomy, whereas one group received a placebo. On micro-computed tomography, in vivo, data showed increased bone volume fraction in the femoral head and shaft areas in the amino-calcium (AC) group. Hematoxylin and eosin staining showed a bone mass and architectural protection in the amino-calcium (AC) group compared with the calcium carbonate and OVX control group. RNA sequencing analysis revealed high expression of osteogenesis-related genes in MC3T3-E1 cells. RNA sequencing revealed a significant fold change in the expression of integrin-binding sialoprotein (IBSP), bone gamma-carboxyglutamate proteins 1 and 2(BGLAP1 and BGLAP2), and periostin (POSTN). The study concluded that supplementing the OVX rats with calcium enhanced bone protection.

Particulate matter 10 exposure affects intestinal functionality in both inflamed 2D intestinal epithelial cell and 3D intestinal organoid models.

Background: A growing body of evidence suggests that particulate matter (PM10) enters the gastrointestinal (GI) tract directly, causing the GI epithelial cells to function less efficiently, leading to inflammation and an imbalance in the gut microbiome. PM10 may, however, act as an exacerbation factor in patients with inflamed intestinal epithelium, which is associated with inflammatory bowel disease. Objective: The purpose of this study was to dissect the pathology mechanism of PM10 exposure in inflamed intestines. Methods: In this study, we established chronically inflamed intestinal epithelium models utilizing two-dimensional (2D) human intestinal epithelial cells (hIECs) and 3D human intestinal organoids (hIOs), which mimic in vivo cellular diversity and function, in order to examine the deleterious effects of PM10 in human intestine-like in vitro models. Results: Inflamed 2D hIECs and 3D hIOs exhibited pathological features, such as inflammation, decreased intestinal markers, and defective epithelial barrier function. In addition, we found that PM10 exposure induced a more severe disturbance of peptide uptake in inflamed 2D hIECs and 3D hIOs than in control cells. This was due to the fact that it interferes with calcium signaling, protein digestion, and absorption pathways. The findings demonstrate that PM10-induced epithelial alterations contribute to the exacerbation of inflammatory disorders caused by the intestine. Conclusions: According to our findings, 2D hIEC and 3D hIO models could be powerful in vitro platforms for the evaluation of the causal relationship between PM exposure and abnormal human intestinal functions.

Mitochondrial matrix protein LETMD1 maintains thermogenic capacity of brown adipose tissue in male mice.

Brown adipose tissue (BAT) has abundant mitochondria with the unique capability of generating heat via uncoupled respiration. Mitochondrial uncoupling protein 1 (UCP1) is activated in BAT during cold stress and dissipates mitochondrial proton motive force generated by the electron transport chain to generate heat. However, other mitochondrial factors required for brown adipocyte respiration and thermogenesis under cold stress are largely unknown. Here, we show LETM1 domain-containing protein 1 (LETMD1) is a BAT-enriched and cold-induced protein required for cold-stimulated respiration and thermogenesis of BAT. Proximity labeling studies reveal that LETMD1 is a mitochondrial matrix protein. Letmd1 knockout male mice display aberrant BAT mitochondria and fail to carry out adaptive thermogenesis under cold stress. Letmd1 knockout BAT is deficient in oxidative phosphorylation (OXPHOS) complex proteins and has impaired mitochondrial respiration. In addition, BAT-specific Letmd1 deficient mice exhibit phenotypes identical to those observed in Letmd1 knockout mice. Collectively, we demonstrate that the BAT-enriched mitochondrial matrix protein LETMD1 plays a tissue-autonomous role that is essential for BAT mitochondrial function and thermogenesis.

Epigenetic regulation of DHRS2 by SUV420H2 inhibits cell apoptosis in renal cell carcinoma.

Renal cell carcinoma (RCC), also known as kidney cancer, is a common malignant tumor of the urinary system. While surgical treatment is essential, novel therapeutic targets and corresponding drugs for RCC are still needed due to the high relapse rate and low five-year survival rate. In this study, we found that SUV420H2 is overexpressed in renal cancers and that high SUV420H2 expression is associated with a poor prognosis, as evidenced by RCC RNA-seq results derived from the TCGA. SUV420H2 knockdown using siRNA led to growth suppression and cell apoptosis in the A498 cell line. Furthermore, we identified DHRS2 as a direct target of SUV420H2 in the apoptosis process through a ChIP assay with a histone 4 lysine 20 (H4K20) trimethylation antibody. Rescue experiments showed that cotreatment with siSUV420H2 and siDHRS2 attenuated cell growth suppression induced by SUV420H2 knockdown only. Additionally, treatment with the SUV420H2 inhibitor A-196 induced cell apoptosis via upregulation of DHRS2. Taken together, our findings suggest that SUV420H2 may be a potential therapeutic target for the treatment of renal cancer.

Epigenetic regulation of SMAD3 by histone methyltransferase SMYD2 promotes lung cancer metastasis.

Epigenetic alterations, especially histone methylation, are key factors in cell migration and invasion in cancer metastasis. However, in lung cancer metastasis, the mechanism by which histone methylation regulates metastasis has not been fully elucidated. Here, we found that the histone methyltransferase SMYD2 is overexpressed in lung cancer and that knockdown of SMYD2 could reduce the rates of cell migration and invasion in lung cancer cell lines via direct downregulation of SMAD3 via SMYD2-mediated epigenetic regulation. Furthermore, using an in vitro epithelial-mesenchymal transition (EMT) system with a Transwell system, we generated highly invasive H1299 (In-H1299) cell lines and observed the suppression of metastatic features by SMYD2 knockdown. Finally, two types of in vivo studies revealed that the formation of metastatic tumors by shSMYD2 was significantly suppressed. Thus, we suggest that SMYD2 is a potential metastasis regulator and that the development of SMYD2-specific inhibitors may help to increase the efficacy of lung cancer treatment.

Tcf7l2 in hepatocytes regulates de novo lipogenesis in diet-induced non-alcoholic fatty liver disease in mice.

AIMS/HYPOTHESIS: Non-alcoholic fatty liver disease (NAFLD) associated with type 2 diabetes may more easily progress towards severe forms of non-alcoholic steatohepatitis (NASH) and cirrhosis. Although the Wnt effector transcription factor 7-like 2 (TCF7L2) is closely associated with type 2 diabetes risk, the role of TCF7L2 in NAFLD development remains unclear. Here, we investigated how changes in TCF7L2 expression in the liver affects hepatic lipid metabolism based on the major risk factors of NAFLD development. METHODS: Tcf7l2 was selectively ablated in the liver of C57BL/6N mice by inducing the albumin (Alb) promoter to recombine Tcf7l2 alleles floxed at exon 5 (liver-specific Tcf7l2-knockout [KO] mice: Alb-Cre;Tcf7l2f/f). Alb-Cre;Tcf7l2f/f and their wild-type (Tcf7l2f/f) littermates were fed a high-fat diet (HFD) or a high-carbohydrate diet (HCD) for 22 weeks to reproduce NAFLD/NASH. Mice were refed a standard chow diet or an HCD to stimulate de novo lipogenesis (DNL) or fed an HFD to provide exogenous fatty acids. We analysed glucose and insulin sensitivity, metabolic respiration, mRNA expression profiles, hepatic triglyceride (TG), hepatic DNL, selected hepatic metabolites, selected plasma metabolites and liver histology. RESULTS: Alb-Cre;Tcf7l2f/f essentially exhibited increased lipogenic genes, but there were no changes in hepatic lipid content in mice fed a normal chow diet. However, following 22 weeks of diet-induced NAFLD/NASH conditions, liver steatosis was exacerbated owing to preferential metabolism of carbohydrate over fat. Indeed, hepatic Tcf7l2 deficiency enhanced liver lipid content in a manner that was dependent on the duration and amount of exposure to carbohydrates, owing to cell-autonomous increases in hepatic DNL. Mechanistically, TCF7L2 regulated the transcriptional activity of Mlxipl (also known as ChREBP) by modulating O-GlcNAcylation and protein content of carbohydrate response element binding protein (ChREBP), and targeted Srebf1 (also called SREBP1) via miRNA (miR)-33-5p in hepatocytes. Eventually, restoring TCF7L2 expression at the physiological level in the liver of Alb-Cre;Tcf7l2f/f mice alleviated liver steatosis without altering body composition under both acute and chronic HCD conditions. CONCLUSIONS/INTERPRETATION: In mice, loss of hepatic Tcf7l2 contributes to liver steatosis by inducing preferential metabolism of carbohydrates via DNL activation. Therefore, TCF7L2 could be a promising regulator of the NAFLD associated with high-carbohydrate diets and diabetes since TCF7L2 deficiency may lead to development of NAFLD by promoting utilisation of excess glucose pools through activating DNL. DATA AVAILABILITY: RNA-sequencing data have been deposited into the NCBI GEO under the accession number GSE162449 ( www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE162449 ).

Characterization of signature trends across the spectrum of non-alcoholic fatty liver disease using deep learning method.

AIMS: The timely diagnosis of different stages in NAFLD is crucial for disease treatment and reversal. We used hepatocellular ballooning to determine different NAFLD stages. MAIN METHODS: We analyzed differentially expressed genes (DEGs) in 78 patients with NAFLD and in healthy controls from previously published RNA-seq data. We identified two expression types in NAFLD progression, calculated the predictive power of candidate genes, and validated them in an independent cohort. We also performed cancer studies with these candidates retrieved from the Cancer Genome Atlas. KEY FINDINGS: We identified 103 DEGs in NAFLD patients compared to healthy controls: 75 genes gradually increased or decreased in the NAFLD stage, whereas 28 genes showed differences only in NASH. The former were enriched in negative regulation and binding-related genes; the latter were involved in positive regulation and cell proliferation. Feature selection showed the gradual up- or down-regulation of 21 genes in NASH compared to controls; 18 were highly expressed only in NASH. Using deep-learning method with subset of features from lasso regression, we obtained reliable determination performance in NAFL and NASH (accuracy: 0.857) and validated these genes using an independent cohort (accuracy: 0.805). From cancer studies, we identified significant differential expression of several candidate genes in LIHC; 5 genes were gradually up-regulated and 6 showing high expression only in NASH were influential to patient survival. SIGNIFICANCE: The identified biomolecular signatures may determine the spectrum of NAFLD and its relationship with HCC, improving clinical diagnosis and prognosis and enabling a therapeutic intervention for NAFLD.

Particulate matter induces ferroptosis by accumulating iron and dysregulating the antioxidant system.

Particulate matter is an air pollutant composed of various components, and has adverse effects on the human body. Particulate matter is known to induce cell death by generating an imbalance in the antioxidant system; however, the underlying mechanism has not been elucidated. In the present study, we demonstrated the cytotoxic effects of the size and composition of particulate matter on small intestine cells. We found that particulate matter 2.5 (PM2.5) with extraction ion (EI) components (PM2.5 EI), is more cytotoxic than PM containing only polycyclic aromatic hydrocarbons (PAHs). Additionally, PM-induced cell death is characteristic of ferroptosis, and includes iron accumulation, lipid peroxidation, and reactive oxygen species (ROS) generation. Furthermore, ferroptosis inhibitor as liproxstatin-1 and iron-chelator as deferiprone attenuated cell mortality, lipid peroxidation, iron accumulation, and ROS production after PM2.5 EI treatment in human small intestinal cells. These results suggest that PM2.5 EI may increase ferroptotic-cell death by iron accumulation and ROS generation, and offer a potential therapeutic clue for inflammatory bowel diseases in human small intestinal cells. [BMB Reports 2023; 56(2): 96-101].

Particulate matter promotes cancer metastasis through increased HBEGF expression in macrophages.

Although many cohort studies have reported that long-term exposure to particulate matter (PM) can cause lung cancer, the molecular mechanisms underlying the PM-induced increase in cancer metastasis remain unclear. To determine whether PM contributes to cancer metastasis, cancer cells were cultured with conditioned medium from PM-treated THP1 cells, and the migration ability of the treated cancer cells was assessed. The key molecules involved were identified using RNA-seq analysis. In addition, metastatic ability was analyzed in vivo by injection of cancer cells into the tail vein and intratracheal injection of PM into the lungs of C57BL/6 mice. We found that PM enhances the expression of heparin-binding EGF-like growth factor (HBEGF) in macrophages, which induces epithelial-to-mesenchymal transition (EMT) in cancer cells, thereby increasing metastasis. Macrophage stimulation by PM results in activation and subsequent nuclear translocation of the aryl hydrocarbon receptor and upregulation of HBEGF. Secreted HBEGF activates EGFR on the cancer cell surface to induce EMT, resulting in increased migration and invasion in vitro and increased metastasis in vivo. Therefore, our study reveals a critical PM-macrophage-cancer cell signaling axis mediating EMT and metastasis and provides an effective therapeutic approach for PM-induced malignancy.

Limosilactobacillus reuteri DS0384 promotes intestinal epithelial maturation via the postbiotic effect in human intestinal organoids and infant mice.

Little is known about the modulatory capacity of the microbiota in early intestinal development. We examined various intestinal models that respond to gut microbial metabolites based on human pluripotent stem cell-derived human intestinal organoids (hIOs): physiologically relevant in vitro fetal-like intestine, intestinal stem cell, and intestinal disease models. We found that a newly isolated Limosilactobacillus reuteri strain DS0384 accelerated maturation of the fetal intestine using 3D hIO with immature fetal characteristics. Comparative metabolomic profiling analysis revealed that the secreted metabolite N-carbamyl glutamic acid (NCG) is involved in the beneficial effect of DS0384 cell-free supernatants on the intestinal maturation of hIOs. Experiments in an intestinal stem cell spheroid model and hIO-based intestinal inflamed model revealed that the cell-free supernatant from DS0384 comprising NCG promoted intestinal stem cell proliferation and was important for intestinal protection against cytokine-induced intestinal epithelial injury. The probiotic properties of DS0384 were also evaluated, including acid and bile tolerance and ability to adhere to human intestinal cells. Seven-day oral administration of DS0384 and cell-free supernatant promoted the intestinal development of newborn mice. Moreover, NCG exerted a protective effect on experimental colitis in mice. These results suggest that DS0384 is a useful agent for probiotic applications and therapeutic treatment for disorders of early gut development and for preventing intestinal barrier dysfunction.

Epigenetic Silencing of CHOP Expression by the Histone Methyltransferase EHMT1 Regulates Apoptosis in Colorectal Cancer Cells.

Colorectal cancer (CRC) has a high mortality rate among cancers worldwide. To reduce this mortality rate, chemotherapy (5-fluorouracil, oxaliplatin, and irinotecan) or targeted therapy (bevacizumab, cetuximab, and panitumumab) has been used to treat CRC. However, due to various side effects and poor responses to CRC treatment, novel therapeutic targets for drug development are needed. In this study, we identified the overexpression of EHMT1 in CRC using RNA sequencing (RNA-seq) data derived from TCGA, and we observed that knocking down EHMT1 expression suppressed cell growth by inducing cell apoptosis in CRC cell lines. In Gene Ontology (GO) term analysis using RNA-seq data, apoptosis-related terms were enriched after EHMT1 knockdown. Moreover, we identified the CHOP gene as a direct target of EHMT1 using a ChIP (chromatin immunoprecipitation) assay with an anti-histone 3 lysine 9 dimethylation (H3K9me2) antibody. Finally, after cotransfection with siEHMT1 and siCHOP, we again confirmed that CHOP-mediated cell apoptosis was induced by EHMT1 knockdown. Our findings reveal that EHMT1 plays a key role in regulating CRC cell apoptosis, suggesting that EHMT1 may be a therapeutic target for the development of cancer inhibitors.

The Comparison of Lower Extremity Length and Angle between Computed Radiography-Based Teleoroentgenogram and EOS® Imaging System.

Background and objectives: The differences between computed radiography-based teleoroentgenograms (CR-based teleoroentgenograms) and an EOS® imaging system were evaluated by measuring lower extremity lengths and alignments. Materials and methods: The leg length [L], femur length [F], tibia length [T], and hip−knee−ankle (HKA) angle were measured in 101 patients with lower extremity disease by a CR-based teleoroentgenogram with computed radiography and an EOS®. The additive length of the femoral and tibial segments (F + T) was determined by adding the two length values. Then, the differences among all five parameters between the two techniques were analyzed. The magnification (mm) was calculated by subtracting the length measurements on the EOS® from those in the scanogram. Furthermore, the magnification percentage (%) was calculated by dividing the magnification with the measurements on the EOS®. Results: The magnification errors (mean ± standard deviation), when comparing both right and left sides, were 7.80 ± 1.41%, 7.3 ± 6.01%, 5.16 ± 1.25%, and 6.45 ± 0.94% for L, F, T, and F + T, respectively. For limb length, the CR-based teleoroentgenogram had an average magnification of 6.8% (range, 5.2 to 7.8%) compared to the EOS® imaging. The two groups displayed a statistical difference (p < 0.01), except for the HKA angle. Conclusions: The CR-based teleoroentgenogram had a magnification of about 6.8% compared to the EOS® imaging system in evaluating lower extremity length. Therefore, more attention must be given to CR-based teleoroentgenograms to correct angular deformities.

Human gut-microbiome-derived propionate coordinates proteasomal degradation via HECTD2 upregulation to target EHMT2 in colorectal cancer.

The human microbiome plays an essential role in the human immune system, food digestion, and protection from harmful bacteria by colonizing the human intestine. Recently, although the human microbiome affects colorectal cancer (CRC) treatment, the mode of action between the microbiome and CRC remains unclear. This study showed that propionate suppressed CRC growth by promoting the proteasomal degradation of euchromatic histone-lysine N-methyltransferase 2 (EHMT2) through HECT domain E3 ubiquitin protein ligase 2 (HECTD2) upregulation. In addition, EHMT2 downregulation reduced the H3K9me2 level on the promoter region of tumor necrosis factor α-induced protein 1 (TNFAIP1) as a novel direct target of EHMT2. Subsequently, TNFAIP1 upregulation induced the apoptosis of CRC cells. Furthermore, using Bacteroides thetaiotaomicron culture medium, we confirmed EHMT2 downregulation via upregulation of HECTD2 and TNFAIP1 upregulation. Finally, we observed the synergistic effect of propionate and an EHMT2 inhibitor (BIX01294) in 3D spheroid culture models. Thus, we suggest the anticancer effects of propionate and EHMT2 as therapeutic targets for colon cancer treatment and may provide the possibility for the synergistic effects of an EHMT2 inhibitor and microbiome in CRC treatment.

Cellular response of lung fibroblasts and epithelial cells to particulate matter10 treatment examined via comparative transcriptome analysis.

Particulate matter (PM) can be categorized by particle size (PM10, PM2.5 and PM1.0), which is an important factor affecting the biological response. Exposure to PM in the air (dust, smoke, dirt and biological contaminants) is clearly associated with lung disease (lung cancer, pneumonia and asthma). Although PM primarily affects lung epithelial cells, the specific response of related cell types to PM remains to be elucidated. The present study performed Gene Ontology (GO) analysis programs (Clustering GO and Database for Annotation, Visualization and Integrated Discovery) on differentially expressed genes in lung epithelial cells (WI­38 VA­13) and fibroblasts (WI­38) following treatment with PM10 and evaluated the cell­specific biological responses related to cell proliferation, apoptosis, adhesion and extracellular matrix production. The results suggested that short­ or long­term exposure to PM may affect cell condition and may consequently be related to several human diseases, including lung cancer and cardiopulmonary disease.

Altered Gene Expression Profiles in Neural Stem Cells Derived from Duchenne Muscular Dystrophy Patients with Intellectual Disability.

Intellectual disability (ID) is a neurodevelopmental disorder defined by below-average intelligence (intelligence quotient of <70) accompanied by adaptive behavior deficits. Defects in the functions of neural stem cells during brain development are closely linked to the pathogenesis of ID. To understand the molecular etiology of ID, we examined neural stem cells from individuals with Duchenne muscular dystrophy (DMD), a genetic disorder in which approximately one-third of the patients exhibit ID. In this study, we generated induced pluripotent stem cells from peripheral blood mononuclear cells from a normal individual and DMD patients with and without ID to identify ID-specific functional and molecular abnormalities. We found defects in neural ectoderm formation in the group of DMD patients with ID. Our transcriptome analysis of patient-derived neural stem cells revealed altered expression of genes related to the hippo signaling pathway and neuroactive ligand-receptor interaction, implicating these in the pathogenesis of ID in patients with DMD.