Luciferase Activity Research Articles

Extracellular vesicle-derived miR-146a as a novel crosstalk mechanism for high-fat induced atherosclerosis by targeting SMAD4

IntroductionExosome-miR-146a is significantly increased in patients with Atherosclerosis (AS), but its mechanism and effect on AS have not been fully elucidated. ObjectivesTo explore the change rule and mechanism of exosomes release, and the role and molecular mechanism of exosome-miR-146a in AS. MethodsWe isolated and identified exosomes from THP-1 macrophages after treating them with ox-LDL. Then used co-immunoprecipitation and silver staining to identify the proteins involved in regulating exosome release. PKH67 was used to label exosomes to confirm that cells can absorb them, and then co-culture with HVSMCs for cell proliferation and migration detection. The target genes of miR-146a were screened and identified through bioinformatics and luciferase activity assay, and the expression of miR-146a and related proteins was detected through qRT-PCR and Western blot in HUVECs. An AS model in LDLR-/- mice induced by a high-fat diet was developed to investigate the impact of exosome-miR-146a on AS. ResultsThe results showed that experimental foam cells from AS showed higher expression of miR-146a. It was observed that NMMHC IIA and HSP70 interacted to regulate the release of exosomes. And HUVECs can absorb exosomes derived from macrophages. In addition, we also found that miR-146a directly targeted the SMAD4 gene to modulate the p38 MAPK signaling pathway, thereby mediating HUVECs damage. Furthermore, exosome-miR-146a induced abnormal proliferation and migration of HVSMCs. The expression of miR-146a was significantly reduced in miR-146a-mimics mice and increased in miR-146a inhibitor mice whereas the inhibition of miR-146a effectively reduced while increasing miR-146a worsened AS in mice. ConclusionOur findings expressed the potential of miR-146a as a favorable therapeutic target for AS, however, further exploration is suggestive for deep understanding of the mechanisms regulating exosome-miR-146a release in vivo and to develop effective therapeutic strategies involving miR-146a.

MicroRNA-650 promotes melanoma metastasis via targeting inhibitor of growth family member 4

ObjectiveThis study aimed to evaluate the effects of microRNA-650 (miR-650) on melanoma metastasis and reveal the regulatory relationship between miR-650 and the inhibitor of growth family member 4 (ING4). MethodsmiR-650 expression was determined in human melanoma WM115 and A-375 cells. WM115 cells were transfected with miR-650 mimic or mimic control. The invasion and migration abilities of transfected WM115 cells were analyzed using Transwell and wound healing assays, respectively. Then, miR-650-overexpression lentivirus vector was constructed and transfected into WM115 cells. After injection into the mice, the number of micro-metastatic foci in the lung tissues was counted. A regulatory relationship between miR-650 and ING4 was identified in WM115 and A-375 cells. ResultsThe miR-650 expression was upregulated in WM115 and A-375 cells. WM115 cells transfected with the miR-650 mimic exhibited higher invasive and migratory abilities than mock cells or cells transfected with negative control (NC). The number of micro-metastatic foci was significantly higher in mice injected with Lenti-miR-650 than that in those injected with mock or NC controls. Transfection with miR-650 mimic observably inhibited the expression of ING4 in WM115 and A-375 cells, whereas transfection with miR-650 inhibitor had the opposite effect. Dual-luciferase reporter gene assay showed that the miR-650 mimic inhibited the luciferase activity of ING4. ConclusionmiR-650 promotes melanoma metastasis by downregulating ING4 expression.

New Smoothened ligands based on the purine scaffold as potential agents for treating pancreatic cancer

Aberrant activation of the Hedgehog (Hh) signalling pathway has been associated with the development and progression of pancreatic cancer. For this reason, blockade of Hh pathway by inhibitors targeting the G protein-coupled receptor Smoothened (SMO) has been considered as a therapeutic target for the treatment of this cancer. In our previous work, we obtained a new SMO ligand based on a purine scaffold (compound I), which showed interesting antitumor activity in several cancer cell lines. In this work, we report the design and synthesis of 17 new purine derivatives, some of which showed high cytotoxic effect on Mia-PaCa-2 (Hh-dependent pancreatic cancer cell lines) and low toxicity on non-neoplastic HEK-293 cells compared with gemcitabine, such as 8f, 8g and 8h (IC50 = 4.56, 4.11 and 3.08 μM, respectively). Two of these purines also showed their ability to bind to SMO through NanoBRET assays (pKi = 5.17 for 8f and 5.01 for 8h), with higher affinities to compound I (pKi = 1.51). In addition, docking studies provided insight the purine substitution pattern is related to the affinity on SMO. Finally, studies of Hh inhibition for selected purines, using a transcriptional functional assay based on luciferase activity in NIH3T3 Shh-Light II cells, demonstrated that 8g reduced GLI activity with a IC50 = 6.4 μM as well as diminished the expression of Hh target genes in two specific Hh-dependent cell models, Med1 cells and Ptch1−/− mouse embryonic fibroblasts. Therefore, our results provide a platform for the design of SMO ligands that could be potential selective cytotoxic agents for the treatment of pancreatic cancer.

Screening of the lncRNA SNHG3 regulating CART expression in the bovine hypothalamus

This study aimed to screen for the long non-coding RNA (lncRNA) small nucleolar RNA host gene 3 (SNHG3) capable of regulating the expression of cocaine- and amphetamine-regulated transcriptional peptide (CART) in the bovine hypothalamus and elucidate the underlying mechanism. StarBase v2.0, NCBI, and DIANA tools were used to predict the lncRNAs targeting miR-381 and miR-491, which were responsible for inhibiting CART expression. The binding sites were analyzed, and the endogenous expression of the selected lncRNAs was determined by semi-quantitative RT-PCR of the hypothalamus tissue from three healthy adult Simmental cows. The dual-luciferase reporter gene assay was employed to detect the targeted binding relationship between miR-381/491 and lncRNAs. The over-expression vectors of lncRNAs, CART, and miR-381/491 mimics were constructed and transfected into 293T cells to reveal the mechanism of lncRNAs in regulating the CART expression. Animal experiments were conducted to analyze the regulatory function of the strongest lncRNA at the cellular level. The results showed that lncRNAs TUG1, SNHG3, H19, SNHG12, and DANCR were expressed in the bovine hypothalamus. The lncRNAs TUG1 and SNHG3 had binding sites for miR-381, and H19, SNHG12, and DANCR had binding sites for miR-491. The dual-luciferase reporter gene assay showed that miR-381 inhibited the relative luciferase activities of TUG1-WT (P < 0.05) and SNHG3-WT (P < 0.01), and miR-491 inhibited the luciferase activities of DANCR-WT (P < 0.05), H19-WT (P < 0.05), and SNHG12-WT (P < 0.01). SNHG3 and SNHG12 up-regulated the CART expression by specifically binding to miR-381 (P < 0.001) and miR-491 (P < 0.01), respectively, and SNHG3 had the strongest effect of regulating CART expression. The results from animal experiments showed that SNHG3 significantly up-regulated the mRNA and protein levels of CART by specifically binding to miR-381. This study confirmed that the lncRNA SNHG3, acting as a competing endogenous RNA of miR-381, significantly up-regulated CART expression at the transcriptional and post-transcriptional levels, laying a foundation for deciphering the mechanism of the molecular network regulation of CART in the bovine hypothalamus.

Correlation between environmental pollutant exposure and cardiopulmonary health by serum biomarker analysis in the Swedish elderly population

ABSTRACT Persistent organic pollutants (POPs) affect human health through the aryl hydrocarbon receptor (AhR) pathway and are implicated in mitochondrial dysfunction. Using data from the PIVUS study, we investigated the associations of serum AhR ligand (POP)-mediated luciferase activity (AhRL), mitochondrial ATP production inhibiting substances (MIS-ATP), and those affecting reactive oxygen species (MIS-ROS) with several metabolic syndrome (MetS) and cardiopulmonary function parameters. These include insulin resistance (HOMA-IR), inflammation, oxidative stress, and cardiopulmonary variables (FVC, FEV1, LV-EF, CCA distensibility). MIS-ATP showed significant correlations with HOMA-IR and pulmonary functions, indicating its direct impact of MIS-ATP on metabolic and pulmonary health. MIS-ROS correlated with oxidative stress markers and CCA distensibility, suggesting a role in systemic inflammatory responses. This study highlights the intricate relationships between environmental pollutant mixture and cardiopulmonary health in MetS as indicated by biomarkers of POP exposure in the elderly population, suggesting POP exposure may influence MetS onset and progression through mitochondrial dysfunction.

Genetic association and functional implications of TLR4 rs1927914 polymorphism on colon cancer risk

BackgroundColon cancer remains a major health concern worldwide, with genetic factors playing a crucial role in its development. Toll-like receptors (TLRs) has been implicated in various cancers, but their role in colon cancer is not well understood. This study aims to identify functional polymorphisms in the promoter and 3′UTR regions of TLRs and evaluate their association with colon cancer susceptibility.MethodsWe conducted a case-control study involving 410 colon cancer patients and 410 healthy controls from the Chinese population. Genotyping of polymorphisms in TLR3, TLR4, TLR5 and TLR7 was performed using PCR-RFLP and TaqMan MGB probes. Using logistic regression analysis, we evaluated the association of TLRs polymorphisms and the susceptibility to colon cancer. To understand the biological implications of the TLR4 rs1927914 polymorphism, we conducted functional assays, including luciferase reporter assay and electrophoretic mobility shift assay (EMSA).ResultsOur results demonstrated that the G-allele of the TLR4 rs1927914 polymorphism is significantly associated with a decreased risk of colon cancer (OR = 0.68, 95%CI = 0.50–0.91). Stratified analysis showed that TLR4 rs1927914 AG or GG genotype contributed to a decreased risk of colon cancer among younger individuals (OR = 0.52, 95%CI = 0.34–0.81), males (OR = 0.58, 95%CI = 0.38–0.87), non-smokers (OR = 0.58, 95%CI = 0.41–0.83) and non-drinker with OR (95%CI) of 0.66 (0.46–0.93). Functional assays demonstrated that in HCT116 and LOVO colon cancer cells, the luciferase activity driven by the TLR4 promoter with the rs1927914A allele was 5.43 and 2.07 times higher, respectively, compared to that driven by the promoter containing the rs1927914G allele. Electrophoretic mobility shift assay (EMSA) results indicated that the rs1927914G allele enhanced transcription factor binding. Using the transcription factor prediction tool, we found that the G allele facilitates binding of the repressive transcription factor Oct1, while the A allele does not.ConclusionThe TLR4 rs1927914 polymorphism influence the susceptibility to colon cancer, with the G allele offering a protective effect through modulation of gene expression. These insights enhance our understanding of the genetic determinants of colon cancer risk and highlight TLR4 as a promising target for cancer prevention strategies.

Macrophage Migration Inhibitory Factor (MIF) Upregulates CXCR7 and Contributes to Chemotherapy Resistance in Colorectal Cancer.

Colorectal cancer is one of the most common malignant tumors worldwide, with high incidence and mortality rates making it a focus of research. Chemotherapy is a primary treatment modality for colon cancer, but chemotherapy resistance severely impacts treatment efficacy. MIF has been found to promote tumor progression and resistance in various cancers. This study aims to investigate the role of MIF in chemotherapy resistance in colon cancer and its potential mechanisms, particularly through the upregulation of CXCR7 expression, affecting the metabolism and drug sensitivity of colon cancer cells. The expression levels of MIF in colon cancer tissues and its association with patient prognosis were evaluated by analyzing TCGA and HPA data. Subsequently, the expression levels of MIF in colon cancer cell lines and resistant cell lines were detected by qRT-PCR and immunohistochemistry, and the effect of MIF on oxaliplatin sensitivity was assessed. The impact of MIF on the metabolic activity of colon cancer cells was measured using a cellular energy metabolism analyzer. Further experiments explored the mechanism by which MIF affects the metabolic activity of colon cancer cells through the upregulation of CXCR7 expression, and the role of CTCF in regulating CXCR7 transcription was validated by silencing CTCF. Finally, the effect of MIF on drug sensitivity of colon cancer cells was verified in a mouse xenograft tumor model. In this study, we found that the expression of MIF in colon cancer tissues was significantly higher than in normal tissues, and high MIF expression was associated with poor prognosis in patients. The expression levels of MIF in resistant colon cancer cell lines were significantly higher than in parental cell lines, and MIF overexpression significantly increased the resistance of colon cancer cells to oxaliplatin. Conversely, silencing MIF significantly reduced the IC50 value of resistant cells and increased apoptosis. MIF overexpression significantly increased the ECAR and OCR levels of colon cancer cells, while MIF knockdown significantly reduced these metabolic indicators. Further studies indicated that MIF affects the metabolic activity of colon cancer cells by upregulating CXCR7 expression. CTCF binding peaks at the CXCR7 promoter region and luciferase activity assays indicated that CTCF regulates CXCR7 transcription, and silencing CTCF significantly enhanced the sensitivity of colon cancer cells to oxaliplatin. In vivo experiments in mice showed that MIF silencing combined with oxaliplatin treatment significantly inhibited tumor growth and increased the necrotic area of tumor tissues. In conclusion, this study reveals the crucial role of MIF in chemotherapy resistance in colon cancer through the upregulation of CXCR7 expression, with CTCF playing an important regulatory role in this process. Our findings provide new theoretical insights and potential therapeutic targets for overcoming chemotherapy resistance in colon cancer. Future research should further explore the roles of MIF and CXCR7 in other types of cancers and the potential of MIF and CXCR7 as therapeutic targets.

CDK5 Upregulated by ELF3 Transcription Promotes IL-1β-induced Inflammation and Extracellular Matrix Degradation in Human Chondrocytes.

Osteoarthritis (OA) is a common chronic disease with age-associated increase in both incidence and prevalence. The cyclin-dependent kinase 5 (CDK5), which is a member of the CDK family, is involved in many chronic diseases. This study was performed to explore the functional role of CDK5 in OA and to discuss the detailed molecular mechanisms. The expressions of CDK5 and ELF3 before or after transfection were detected with reverse transcription-quantitative PCR (RT-qPCR) and western blot. 5-ethynyl-2'-deoxyuridine (Edu) and terminal deoxynucleoitidyl transferase-mediated nick-end labeling (TUNEL) assays were used to detect the proliferation and apoptosis of C28/I2 cells. The levels of inflammatory cytokines were estimated using enzyme-linked immunosorbent assay (ELISA) while the expressions of proteins implicated in extracellular matrix (ECM) degradation- and apoptosis were detected using western blot. Additionally, the activity of CDK5 promoters and its binding with ELF3 were detected using luciferase activity assay and chromatin immunoprecipitation (CHIP) assay. In the present study, it was discovered that the mRNA and protein expressions of CDK5 were significantly increased in IL-1β-induced C28/I2 cells. After depleting CDK5 expression, the apoptosis, inflammation and ECM in C28/I2 cells with IL-1β induction were suppressed. It was also found that ELF3 expression was increased in IL-1β-induced C28/I2 cells and acted as a transcription factor binding to the CDK5 promoter to regulate its transcriptional expression. The further experiments evidenced that ELF3 overexpression partially reversed the inhibitory effects of CDK5 deficiency on IL-1β-induced apoptosis, inflammation and ECM in C28/I2 cells. Collectively, CDK5 that upregulated by ELF3 transcription could promote the development of OA.

Evaluation of a Novel Lateral Emitting Laser Fiber for Near-Infrared Photoimmunotherapy.

Near-infrared photoimmunotherapy (NIR-PIT) is a new cancer therapy that uses NIR light and conjugates of a tumor-targeting monoclonal antibody and phthalocyanine dye. In clinical practice, frontal and cylindrical diffusers are the only options for NIR illumination. However, illumination in a narrow space is technically difficult with such diffusers. Therefore, we evaluated a lateral illumination system using a lateral emitting laser (LEL) fiber. The LEL fiber illuminated a certain area in a lateral direction. NIR-PIT with an LEL fiber reduced luciferase activity in a light-dose-dependent manner in A431-GFP-luc cells in vitro and significantly suppressed tumor proliferation in a xenograft mouse model. To evaluate the usefulness of the LEL fiber in the illumination of a narrow space, a tumor was illuminated from the inside of a cylinder, mimicking a narrow space, and the fluorescence intensity in the tumor was monitored. In the frontal diffuser, NIR light was unevenly delivered and little light reached a distal tumor area from the illuminated side. By contrast, the LEL fiber allowed a uniform illumination of the entire tumor, and a loss of fluorescence was observed even in distal areas. These findings suggested that the LEL fiber can be used for NIR-PIT and is suitable for NIR light illumination in a narrow space.

Bezafibrate mitigates oxidized-low density lipoprotein (ox-LDL)-induced the attachment of monocytes to endothelial cells: An implication in atherosclerosis.

Oxidized forms of low-density lipoproteins (ox-LDL)-associated endothelial dysfunction and subsequent monocyte adhesion play an important role in the development of atherosclerosis (AS). Bezafibrate (BEZ) is a peroxisome proliferator-activated receptor (pan-PPAR) agonist licensed as a hypolipidemic drug. However, the effects of BEZ on endothelial dysfunction are less reported. In this study, we aim to investigate the protective effects of BEZ on ox-LDL-challenged vascular endothelial cells to evaluate its potential value in treating AS. Human aortic endothelial cells (HAECs) and THP-1 cells were used to establish an In Vitro AS model. Cell Counting Kit-8 (CCK-8) assay, Real-time PCR, Western blot analysis, and Enzyme-linked immunosorbent assay (ELISA) were used to test the data. As expected, treatment with BEZ suppressed the expression of vascular endothelial growth factor A (VEGF-A), tissue factor (TF), Interleukin 12 (IL-12), tumor necrosis factor (TNF-α), and monocyte chemoattractant protein-1 (MCP-1). BEZ was also found to inhibit ox-LDL-induced expression of the endothelial adhesion molecules vascular cellular adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) in HAECs. Correspondingly, BEZ prevented attachment of THP-1 monocytes to ox-LDL-incubated HAECs. Mechanically, BEZ was found to prevent NF-κB activation by reducing the levels of nuclear NF-κB p65 and inhibiting luciferase activity of NF-κB. Our study revealed the pharmacological function of BEZ in protecting endothelial dysfunction against ox-LDL, which may provide valuable insight for the clinical application of BEZ.

A class of chemical compounds enhances clustering of muscle nicotinic acetylcholine receptor in cultured myogenic cells

Neuromuscular signal transmission is affected in various diseases including myasthenia gravis, congenital myasthenic syndromes, and sarcopenia. We used an ATF2-luciferase system to monitor the phosphorylation of MuSK in HEK293 cells introduced with MUSK and LRP4 cDNAs to find novel chemical compounds that enhanced agrin-mediated acetylcholine receptor (AChR) clustering. Four compounds with similar chemical structures carrying benzene rings and heterocyclic rings increased the luciferase activities 8- to 30-folds, and two of them showed continuously graded dose dependence. The effects were higher than that of disulfiram, a clinically available aldehyde dehydrogenase inhibitor, which we identified to be the most competent preapproved drug to enhance ATF2-luciferase activity in the same assay system. In C2C12 myotubes, all the compounds increased the area, intensity, length, and number of AChR clusters. Three of the four compounds increased the phosphorylation of MuSK, but not of Dok7, JNK. ERK, or p38. Monitoring cell toxicity using the neurite elongation of NSC34 neuronal cells as a surrogate marker showed that all the compounds had no effects on the neurite elongation up to 1 μM. Extensive docking simulation and binding structure prediction of the four compounds with all available human proteins using AutoDock Vina and DiffDock showed that the four compounds were unlikely to directly bind to MuSK or Dok7, and the exact target remained unknown. The identified compounds are expected to serve as a seed to develop a novel therapeutic agent to treat defective NMJ signal transmission.

LncRNA PITPNA-AS1 mediates the diagnostic potential of miR-129-5p in prostate cancer

BackgroundLncRNA has an effective value in many diseases, which has long been applied in the diagnosis, treatment and prognosis of prostate cancer. This study focused on lncRNA PITPNA-AS1, and its diagnostic potential in prostate cancer has been explored.MethodsThe expression of PITPNA-AS1 and miR-129-5p in prostate cancer serum and sample cells was determined by real-time quantitative polymerase chain reaction (RT-qPCR). The relationship between the expression of PITPNA-AS1 and clinicopathological parameters was considered. ROC curve prompted the diagnostic value of PITPNA-AS1. The effect of PITPNA-AS1 on prostate cancer cells was verified using vitro cells assay. Luciferase activity assay and RIP assay demonstrated the sponge relationship of PITPNA-AS1 to miR-129-5p.ResultsPITPNA-AS1 level was increased, while miR-129-5p was obviously decreased in prostate cancer. PITPNA-AS1 expression was associated with Gleason grade, lymph node metastasis and TNM stage in patients. The area under the curve (AUC) was 0.910, with high sensitivity and specificity. PITPNA-AS1 was elucidated to directly target miR-129-5p, whereas silencing PITPNA-AS1 negatively affected prostate cancer cell proliferation, migration and invasion. Intervention of miR-129-5p inhibitor reversed the effect of silencing PITPNA-AS1 on cells.ConclusionsPITPNA-AS1 was relatively highly expressed in prostate cancer and mediated the pathophysiological process of patients, which may serve as a diagnostic indicator. Silencing of the PITPNA-AS1 sponge miR-129-5p inhibited the biological function of the cells, indicating that PITPNA-AS1 may represent a novel therapeutic target for prostate cancer.

LncRNAH19 acts as a ceRNA of let-7 g to facilitate endothelial-to-mesenchymal transition in hypoxic pulmonary hypertension via regulating TGF-β signalling pathway

BackgroundHypoxic pulmonary hypertension (HPH) is a challenging lung arterial disorder with remarkably high incidence and mortality rates, and the efficiency of current HPH treatment strategies is unsatisfactory. Endothelial-to-mesenchymal transition (EndMT) in the pulmonary artery plays a crucial role in HPH. Previous studies have shown that lncRNA-H19 (H19) is involved in many cardiovascular diseases by regulating cell proliferation and differentiation but the role of H19 in EndMT in HPH has not been defined.MethodsIn this research, the expression of H19 was investigated in PAH human patients and rat models. Then, we established a hypoxia-induced HPH rat model to evaluate H19 function in HPH by Echocardiography and hemodynamic measurements. Moreover, luciferase reporter gene detection, and western blotting were used to explore the mechanism of H19.ResultsHere, we first found that the expression of H19 was significantly increased in the endodermis of pulmonary arteries and that H19 deficiency obviously ameliorated pulmonary vascular remodelling and right heart failure in HPH rats, and these effects were associated with inhibition of EndMT. Moreover, an analysis of luciferase activity indicated that microRNA-let-7 g (let-7 g) was a direct target of H19. H19 deficiency or let-7 g overexpression can markedly downregulate the expression of TGFβR1, a novel target gene of let-7 g. Furthermore, inhibition of TGFβR1 induced similar effects to H19 deficiency.ConclusionsIn summary, our findings demonstrate that the H19/let-7 g/TGFβR1 axis is crucial in the pathogenesis of HPH by stimulating EndMT. Our study may provide new ideas for further research on HPH therapy in the near future.

IPSC-NSCs-derived exosomal let-7b-5p improves motor function after spinal cord Injury by modulating microglial/macrophage pyroptosis

BackgroundFollowing spinal cord injury (SCI), the inflammatory storm initiated by microglia/macrophages poses a significant impediment to the recovery process. Exosomes play a crucial role in the transport of miRNAs, facilitating essential cellular communication through the transfer of genetic material. However, the miRNAs from iPSC-NSCs-Exos and their potential mechanisms leading to repair after SCI remain unclear. This study aims to explore the role of iPSC-NSCs-Exos in microglia/macrophage pyroptosis and reveal their potential mechanisms.MethodsiPSC-NSCs-Exos were characterized and identified using transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and Western blot. A mouse SCI model and a series of in vivo and in vitro experiments were conducted to investigate the therapeutic effects of iPSC-NSCs-Exos. Subsequently, miRNA microarray analysis and rescue experiments were performed to confirm the role of miRNAs in iPSC-NSCs-Exos in SCI. Mechanistic studies were carried out using Western blot, luciferase activity assays, and RNA-ChIP.ResultsOur findings revealed that iPSC-NSCs-derived exosomes inhibited microglia/macrophage pyroptosis at 7 days post-SCI, maintaining myelin integrity and promoting axonal growth, ultimately improving mice motor function. The miRNA microarray showed let-7b-5p to be highly enriched in iPSC-NSCs-Exos, and LRIG3 was identified as the target gene of let-7b-5p. Through a series of rescue experiments, we uncovered the connection between iPSC-NSCs and microglia/macrophages, revealing a novel target for treating SCI.ConclusionIn conclusion, we discovered that iPSC-NSCs-derived exosomes can package and deliver let-7b-5p, regulating the expression of LRIG3 to ameliorate microglia/macrophage pyroptosis and enhance motor function in mice after SCI. This highlights the potential of combined therapy with iPSC-NSCs-Exos and let-7b-5p in promoting functional recovery and limiting inflammation following SCI.

Identification of new benzofuran derivatives as STING agonists with broad-spectrum antiviral activity

The Stimulator of Interferon Genes (STING) is involved in cytosolic DNA sensing and type I Interferons (IFN-I) induction. Aiming to identify new STING agonists with antiviral activity and given the known biological activity of benzothiazole and benzimidazole derivatives, a series of benzofuran derivatives were tested for their ability to act as STING agonists, induce IFN-I and inhibit viral replication. Compounds were firstly evaluated in a gene reporter assay measuring luciferase activity driven by the human IFN-β promoter in cells expressing exogenous STING (HEK293T). Seven of them were able to induce IFN-β transcription while no induction of the IFN promoter was observed in the presence of a mutated and inactive STING, showing specific protein-ligand interaction. Docking studies were performed to predict their putative binding mode. The best hit compounds were then tested on human coronavirus 229E replication in BEAS-2B and MRC-5 cells and three derivatives showed EC50 values in the μM range. Such compounds were also tested on SARS-CoV-2 replication in BEAS-2B cells and in Calu-3 showing they can inhibit SARS-CoV-2 replication at nanomolar concentrations. To further confirm their IFN-dependent antiviral activity, compounds were tested to verify their effect on phospho-IRF3 nuclear localization, that was found to be induced by benzofuran derivatives, and SARS-CoV-2 replication in Vero E6 cells, lacking IFN production, founding them to be inactive. In conclusion, we identified benzofurans as STING-dependent immunostimulatory compounds and host-targeting inhibitors of coronaviruses representing a novel chemical scaffold for the development of broad-spectrum antivirals.

The role and mechanism of action of miR-483-3p in mediating the effects of IGF-1 on human renal tubular epithelial cells induced by high glucose

This study aimed to elucidate the influence of miR-483-3p on human renal tubular epithelial cells (HK-2) under high glucose conditions and to understand its mechanism. Human proximal tubular epithelial cells (HK-2) were exposed to 50 mmol/L glucose for 48 h to establish a renal tubular epithelial cell injury model, denoted as the high glucose group (HG group). Cells were also cultured for 48 h in a medium containing 5.5 mmol/L glucose, serving as the low glucose group. Transfection was performed in various groups: HK-2 + low glucose (control group), high glucose (50 mM) (HG group), high glucose + miR-483-3p mimics (HG + mimics group), high glucose +miR-483-3p inhibitor (HG + inhibitor group), and corresponding negative controls. Real-time quantitative polymerase chain reaction (qPCR) assessed the mRNA expression of miR-483-3p, bax, bcl-2, and caspase-3. Western blot determined the corresponding protein levels. Proliferation was assessed using the CCK-8 assay, and cell apoptosis was analyzed using the fluorescence TUNEL method. Western blot and Masson’s staining were conducted to observe alterations in cell fibrosis post miR-483-3p transfection. Furthermore, a dual-luciferase assay investigated the targeting relationship between miR-483-3p and IGF-1. The CCK8 assay demonstrated that the HG + mimics group inhibited HK-2 cell proliferation, while the fluorescent TUNEL method revealed induced cell apoptosis in this group. Conversely, the HG + inhibitor group promoted cell proliferation and suppressed cell apoptosis. The HG + mimics group upregulated mRNA and protein expression of pro-apoptotic markers (bax and caspase-3), while downregulating anti-apoptotic marker (bcl-2) expression. In contrast, the HG + inhibitor group showed opposite effects. Collagen I and FN protein levels were significantly elevated in the HG + mimics group compared to controls (P < 0.05). Conversely, in the HG + inhibitor group, the protein expression of Collagen I and FN was notably reduced compared to the HG group (P < 0.05). The dual luciferase reporter assay confirmed that miR-483-3p could inhibit the luciferase activity of IGF-1’s 3′-UTR region (P < 0.05). miR-483-3p exerts targeted regulation on IGF-1, promoting apoptosis and fibrosis in renal tubular epithelial cells induced by high glucose conditions.

2,3,5,4′-tetrahydroxy-stilbene-2-O-β-D-glucoside promotes liver regeneration after partial hepatectomy in mice: The potential involvement of PPARα-mediated fatty acid metabolism

Ethnopharmacological relevance2,3,5,4′-tetrahydroxy-stilbene-2-O-β-D-glucoside (TSG) is the principal bioactive compound contained in Polygonum multiflorum Thunb. (PMT), which is traditionally recorded to possess tonic and anti-aging efficacy. Aim of the studyTo identify the TSG-provided promotion on liver regeneration (LR) following partial hepatectomy (PHx) in mice and to explicate its involved mechanism. Materials and methodsThe promotion of TSG on LR was evaluated by hematoxylin and eosin (H&E), 5-bromodeoxyuridinc (BrdU) and Ki-67 staining, and measuring the level of proliferating cell nuclear antigen (PCNA) and Cyclin D1 in mice with PHx at different time points. Gene Expression Omnibus (GEO, GSE15239) database and the label-free quantitative proteomics from liver of mice at 24 h after PHx were integrated to identify potential involved critical proteins, which were verified by Western-blot, Real-time polymerase chain reaction (RT-PCR), molecular docking and luciferase activity assay. Primary hepatocytes isolated from mice were used to investigate the TSG-provided promotion on proliferation in vitro. ResultsTSG (20 mg/kg) promoted LR in mice after PHx. Results from RNA expression data from clinical samples and proteomic analysis from liver tissues indicated that peroxisome proliferator-activated receptor α (PPARα)-mediated fatty acid metabolism pathway were crucially associated with the TSG-provided promotion on LR. TSG enhanced the nuclear translocation of PPARα and the mRNA expression of a series of PPARα-regulated downstream genes. In addition, TSG lowered hepatic triglyceride (TG) and non-esterified fatty acid (NEFA) amounts and increased hepatic adenosine triphosphate (ATP) level in mice after PHx. TSG up-regulated the transcriptional activity of PPARα in vitro. Next results displayed that TSG promoted cell proliferation as well as ATP level in mice primary hepatocytes, which were abolished when PPARα was suppressed. Meanwhile, the cell viability was also elevated in mice primary hepatocytes treated with ATP. ConclusionActivating PPARα-mediated fatty acid β-oxidation (FAO) pathway led to the production of ATP, which contributed to the TSG-provided promotion on LR after PHx in mice.

Functional Impact of the FADS1 rs174546 Single Nucleotide Polymorphism on Serum Lipid Levels: Insights from Molecular Mechanisms and Therapeutic Perspectives in Korean Population.

Single nucleotide polymorphisms (SNP) in the fatty acid desaturase 1 (FADS1) gene is suggested as risk factor of metabolic diseases in genome-wide association studies (GWAS). This study hypothesized that FADS1_rs174546T associates with serum triglycerides (TG) in Korean Genome and Epidemiology Study (KoGES). In addition, functional study of SNP genotypes in cultured cells is performed. FADS1_rs174546T is associated with high level of serum TG (effect size of variant: 6.48 ± 1.84mgdL-1) in Korean individuals (normotriglyceridemia, n = 5128; hypertriglyceridemia, n = 3714). Functional study in cells with FADS1_rs174546T, shows reduced transcriptional activity, when compared with rs174546C. MiR-6728-3p, which is predicted to bind with rs174546T, decreases transcriptional activity of rs174546T but not in rs174546C, and it is reversed by miR-6728-3p inhibitor. Formononetin is selected as binding molecule to 3'-UTR of FADS1 and increases luciferase activity in both rs174546 (C/T). Moreover, formononetin compensates for the reduced luciferase activity by rs174546T and miR-6728-3p. Formononetin also increases endogenous FADS1 expression and long-chain polyunsaturated fatty acid (LC-PUFA) ratio. FADS1_rs174546T is a crucial risk factor for hypertriglyceridemia in the Koreans potentially through the interaction with miR-6728-3p. Formononetin can be a potent dietary intervention to prevent and improve hypertriglyceridemia in both rs174546 (C/T) populations.

LILRB4 on multiple myeloma cells promotes bone lesion by p-SHP2/NF-κB/RELT signal pathway

BackgroundLeukocyte Ig-like receptor B family 4 (LILRB4) as an immune checkpoint on myeloid cells is a potential target for tumor therapy. Extensive osteolytic bone lesion is the most characteristic feature of multiple myeloma. It is unclear whether ectopic LILRB4 on multiple myeloma regulates bone lesion.MethodsThe conditioned medium (CM) from LILRB4-WT and -KO cells was used to analyze the effects of LILRB4 on osteoclasts and osteoblasts. Xenograft, syngeneic and patient derived xenograft models were constructed, and micro-CT, H&E staining were used to observe the bone lesion. RNA-seq, cytokine array, qPCR, the activity of luciferase, Co-IP and western blotting were used to clarify the mechanism by which LILRB4 mediated bone damage in multiple myeloma.ResultsWe comprehensively analyzed the expression of LILRB4 in various tumor tissue arrays, and found that LILRB4 was highly expressed in multiple myeloma samples. The patient’s imaging data showed that the higher the expression level of LILRB4, the more serious the bone lesion in patients with multiple myeloma. The conditioned medium from LILRB4-WT not -KO cells could significantly promote the differentiation and maturation of osteoclasts. Xenograft, syngeneic and patient derived xenograft models furtherly confirmed that LILRB4 could mediate bone lesion of multiple myeloma. Next, cytokine array was performed to identify the differentially expressed cytokines, and RELT was identified and regulated by LILRB4. The overexpression or exogenous RELT could regenerate the bone damage in LILRB4-KO cells in vitro and in vivo. The deletion of LILRB4, anti-LILRB4 alone or in combination with bortezomib could significantly delay the progression of bone lesion of multiple myeloma.ConclusionsOur findings indicated that LILRB4 promoted the bone lesion by promoting the differentiation and mature of osteoclasts through secreting RELT, and blocking LILRB4 singling pathway could inhibit the bone lesion.

Mechanism of BRD4 Inhibitor-Mediated c-MYC Expression and Regulation of AR Expression to Inhibit Prostate Cancer

Prostate cancer is a major health challenge, probably because of regulatory role of hormones that has not been clearly studied. Our study explored BRD4’s role in the development of prostate cancer. BRD4 expression was detected in tumor tissues by RT-PCR method. Transwell detected cell function after different BRD4 expressions, while Target scan detected the relationship between c-MYC and BRD4, including protein expression between the two luciferase activity and Western-blot. Western-blot detected the protein expression after addition of c-MYC, and further verified the effect of c-MYC expression on AR. We proved that, BRD4 was highly expressed in tumor tissues, and inhibiting BRD4 expression significantly inhibited tumor cell invasion and proliferation. BRD4 targets negative feedback to regulate the expression of c-MYC. Further results showed that, addition of BRD4 activated c-MYC signal transduction and inhibited prostate cancer development. Our study reveals that, BRD4 regulates AR to inhibit prostate cancer by regulating c-MYC. BRD4 is involved in AR-mediated regulation of PCa cells. In addition, inhibiting the expression of BRD4 can inhibit pathological progression of prostate cancer cells.