Cell Migration Capacity Research Articles

Natural killer cell effector function is critical for host defense against alcohol-associated bacterial pneumonia

Alcohol use is an independent risk factor for the development of bacterial pneumonia due, in part, to impaired mucus-facilitated clearance, macrophage phagocytosis, and recruitment of neutrophils. Alcohol consumption is also known to reduce peripheral natural killer (NK) cell numbers and compromise NK cell cytolytic activity, especially NK cells with a mature phenotype. However, the role of innate lymphocytes, such as NK cells during host defense against alcohol-associated bacterial pneumonia is essentially unknown. We have previously shown that indole supplementation mitigates increases in pulmonary bacterial burden and improves pulmonary NK cell recruitment in alcohol-fed mice, which were dependent on aryl hydrocarbon receptor (AhR) signaling. Employing a binge-on-chronic alcohol-feeding model we sought to define the role and interaction of indole and NK cells during pulmonary host defense against alcohol-associated pneumonia. We demonstrate that alcohol dysregulates NK cell effector function and pulmonary recruitment via alterations in two key signaling pathways. We found that alcohol increases transforming growth factor beta (TGF-β) signaling while suppressing AhR signaling. We further demonstrated that NK cells isolated from alcohol-fed mice have a reduced ability to kill Klebsiella pneumoniae. NK cell migratory capacity to chemokines was also significantly altered by alcohol, as NK cells isolated from alcohol-fed mice exhibited preferential migration in response to CXCR3 chemokines but exhibited reduced migration in response to CCR2, CXCR4, and CX3CR1 chemokines. Together this data suggests that alcohol disrupts NK cell-specific TGF-β and AhR signaling pathways leading to decreased pulmonary recruitment and cytolytic activity thereby increasing susceptibility to alcohol-associated bacterial pneumonia.

Effect of platelet-rich fibrin and concentrated growth factor on the regenerative potential of human-induced pluripotent stem cells: A comparative analysis.

Platelet-rich fibrin (PRF) has been used, while concentrated growth factor (CGF) has recently evolved as a bioscaffold in regenerative endodontics. This study aimed to evaluate the effect of PRF and CGF on the proliferation, migration, and differentiation of human-induced pluripotent stem cells (hiPSCs). CGF and PRF were fabricated from voluntarily donated human blood, and a conditioned medium was prepared. HiPSCs were isolated and cultivated on a conditioned medium for 12 days. The proliferation rate was analyzed using a trypan blue assay on days 9, 10, and 11. The migratory rate was evaluated using a wound healing assay after 24, 48, and 72 h. For assessing the differentiation of hiPSCs, various markers with quantitative real-time polymerase chain reactions on day 12 were used. Mesenchymal phenotypic transition was seen with an increase in proliferation rate in the PRF group more than in the CGF group on day 9, along with the differentiation of cells with an increase in osteoblastic markers on day 12 in both groups. The migratory capacity of cells was significantly increased in the CGF and PRF groups, with a greater increase in the CGF group. CGF and PRF extend the duration of growth factor activity and enhance cell proliferation and osteogenic differentiation, with hiPSCs serving as a bioscaffold with high regenerative potential.

GGT5 as a promising prognostic biomarker and its effects on tumor cell progression in gastric cancer.

Gastric cancer (GC) is a gastric malignant tumor with over 1 million new cases globally each year. There are many diagnostic methods for GC, but due to the hidden early symptoms of GC, early GC is easy to be missed and misdiagnosed, which affects the follow-up treatment of patients. The early and accurate diagnosis of GC is of great significance for the treatment and survival of GC patients. Our laboratory study found that gamma-glutamyl transferase (GGT) was highly expressed in GC patients, but the mechanism of GGT family genes in the occurrence and development of GC remained to be further studied. Therefore, this study aimed to explore the mechanism of GGT family functional gene GGT5 regulating the proliferation and migration of GC cells, and provide a possible new biomarker for the early diagnosis of GC. The value of serum GGT in GC patients was first statistically analyzed. Then, The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) datasets were used to analyze the mRNA expression of GGT5 in GC, and its clinical relationship and function. Furthermore, expression of GGT5 was reduced by lentivirus RNA interference and verified by polymerase chain reaction (PCR), Cell Counting Kit-8 (CCK-8) and 5-ethynyl-2'-deoxyuridine (EdU) assays were used to detect cell proliferation after GGT5 knockdown. Scratch and Transwell assays were applied to observe cell migration after knockdown of GGT5. Finally, Western blot assays were observed to demonstrate PI3K/AKT-MAPK and MMPs expression levels after knockdown of GGT5. Serum GGT was expressed at a high level in GC patients. GGT5 was highly expressed in GC tissues, and was associated with poor prognosis and clinical stage of GC. GGT5 might be involved in the regulation of vascular development and angiogenesis, as well as in the mechanisms of cell motility and migration, and it was positively correlated with the PI3K/AKT pathway. The proliferation and migration capacity of GC cells was dampened by downregulation of GGT5. GGT5 mediated proliferation and migration of GC cells by directly targeting PI3K/AKT-MAPK-MMPs pathways. Low expression of GGT5 reduced proliferation and migration in GC cells by modulating the PI3K/AKT-MAPK-MMPs pathway, and GGT5 might be a new target for GC.

Cytotoxicity and cell migration evaluation of a strontium silicate-based root canal sealer on stem cells from rat apical papilla: an in vitro study

BackgroundCalcium silicate-based bioceramics have been applied in endodontics as advantageous materials for years, many chemical components and new synthesizing methods were used to improve the base formulation of the materials for positively affecting the sealers properties. Recently, a novel biomaterial formulation, grounded in strontium silicate, has been introduced to the market, offering potential advancements in the field.ObjectiveTo comparatively analyze the cytotoxicity and cell migration effects of a novel strontium silicate-based bioceramic material (CRoot SP) and those of calcium silicate-based (iRoot SP) and epoxide amine resin (AH Plus) sealers on stem cells derived from rat apical papilla(rSCAPs).MethodsrSCAPs were isolated and characterized in vitro and subsequently cultured in the presence of various concentrations of CRoot SP, iRoot SP and AH Plus extracts. Cytotoxicity was assessed by CCK-8 assay, and cell-migration capacity was assessed by using wound healing assays .ResultsNo significant differences in cell viability were observed in the 0.02 mg/mL and 0.2 mg/mL sealer groups. The cell viability of CRoot SP was consistently greater than that of iRoot SP at concentrations of 5 mg/mL and 10 mg/mL across all time points. Maximum cytotoxic effect was noted on day 5 with 10 mg/mL AH Plus.The scratch was partly healed by cell migration in all groups at 24 h, and the 0.02 mg/mL, and 0.2 mg/mL CRoot SP exerted beneficial effects on rSCAPs migration.ConclusionsCRoot SP exhibited less cytotoxic than the iRoot SP and AH Plus extracts after setting. A lower concentration of CRoot SP thus promotes the cell migration capacity of rSCAPs, and it may achieve better tissue repair during root canal treatment.

WYC-209 suppresses gastric cancer by down-regulating FGF18 via inactivating the STAT3 signaling pathway

BackgroundGastric cancer (GC) is regarded as a major health burden all over the world. WYC-209 inhibits the growth and metastasis of tumor-repopulating cells (TRCs). However, its effectiveness on GC was unexplored. Herein, this study aims to investigate the effect of WYC-209 on GC and elucidate its underlying mechanism. MethodsWe examined the effects of WYC-209 on cell survival, migration, invasion, and colony-forming capacities of two GC cell lines (AGS and HGC-27). Subsequently, RNA-seq and enrichment analyses were performed to screen the differentially expressed genes (DEGs) and the enriched signaling pathways. To further explore the underlying mechanism, loss- and gain-function experiments, Chromatin immunoprecipitation, and luciferase reporter were conducted. Finally, xenograft models were constructed to examine the effects of WYC-209 in vivo. ResultsWYC-209 significantly inhibited cell motility in vitro and tumor growth in vivo. RNA-seq performed in AGS cells after WYC-209 treatment revealed that the inhibition effect of WYC-209 on GCs may be associated with the down-regulation of fibroblast growth factor-18 (FGF18), and pleasantly, FGF18 overexpression abrogated the suppression effect of the drug. In addition, we found that WYC-209 attenuated the activation of the Signal Transducer and Activator of Transcription 3 (STAT3) signaling pathway, and impeded the FGF18 levels expressed in GCs. Importantly, the WYC-209 treatment circumvented the binding of STAT3 to the FGF18 promoter, suggested that WYC-209 down-regulated FGF18 expression via the STAT3 signaling pathway. ConclusionTogether, our findings presented the promise of WYC-209 in suppressing GC by down-regulating FGF18 expression through inactivating the STAT3 signaling pathway.

The novel circFKBP8/miR-432-5p/E2F7 cascade functions as a regulatory network in breast cancer

BackgroundCircular RNAs (circRNAs) are capable of affecting breast cancer (BC) development. However, the role and underneath mechanism of circFKBP8 (also known as hsa_circ_0000915) in BC remain largely unknown.MethodsExpression analyses were performed using quantitative real-time polymerase chain reaction (qRT-PCR), western blot, and immunohistochemistry (IHC) assays. Effects on cell functional phenotypes were determined by assessing cell proliferation, migratory capacity, invasion, and stemness in vitro. The relationship between microRNA (miR)-432-5p and circFKBP8 or E2F transcription factor 7 (E2F7) was examined by RNA pull-down, dual-luciferase reporter, and RNA immunoprecipitation (RIP) assays. Xenograft assays were used to identify the function of circFKBP8 in vivo.ResultsCircFKBP8 was presented at high levels in BC tissues and cells. High circFKBP8 expression was associated with worse overall survival in BC patients. CircFKBP8 suppression inhibited BC cell proliferation, migratory capacity, invasion and stemness in vitro. CircFKBP8 suppression blocked xenograft tumor growth in vivo. Mechanistically, circFKBP8 functioned as a miR-432-5p sponge to modulate E2F7 expression. CircFKBP8 modulated BC cell malignant behaviors by miR-432-5p, and miR-432-5p affected these cell phenotypes through E2F7.ConclusionOur observations prove that circFKBP8 promotes BC malignant phenotypes through the miR-432-5p/E2F7 cascade, offering a promising therapeutic and prognostic target for BC.

Base-Mediated Chemodivergent [4 + 1] and [2 + 1] Cycloadditions of N-Alkylpyridiniums and Enones.

Divergent synthesis of structurally different products from the same kinds of starting materials is highly synthetically useful but very challenging. Herein, we reported a base-mediated chemodivergent [4 + 1] and [2 + 1] cycloaddition of N-alkylpyridinium and enone under mild conditions, leading to furan-fused bicycles with high diastereoselectivity and spirobicycles, respectively, from moderate to high yields. N-Alkylpyridinium salts were modular nucleophilic transfer reagents and C1 synthons, which underwent tandem Michael addition to the α,β-unsaturated ketones and cyclization under the base conditions. Late-stage derivatization of 4-propyldicyclohexylanone from an important industrial raw of liquid crystal display (LCD) screens was realized. In vitro, compound 3f exhibited good activities against human colon cancer cells (HCT116) with IC50 values in 9.82 ± 0.27 μM. Further biological evaluations investigated the mechanism of the effective inhibition of cell growth, including apoptosis ratio detection, cell cycle analysis, and migration capacity of HCT116 cells. In apoptosis effect studies, complex 3f increased the percentage of apoptotic HCT116 cells to 26.8% (15 μM).

Axonal Growth and Fasciculation of Spinal Neurons Promoted by Aldynoglia in Alkaline Fibrin Hydrogel: Influence of Tol-51 Sulfoglycolipid.

Traumatic spinal cord injury (tSCI) has complex pathophysiological events that begin after the initial trauma. One such event is fibroglial scar formation by fibroblasts and reactive astrocytes. A strong inhibition of axonal growth is caused by the activated astroglial cells as a component of fibroglial scarring through the production of inhibitory molecules, such as chondroitin sulfate proteoglycans or myelin-associated proteins. Here, we used neural precursor cells (aldynoglia) as promoters of axonal growth and a fibrin hydrogel gelled under alkaline conditions to support and guide neuronal cell growth, respectively. We added Tol-51 sulfoglycolipid as a synthetic inhibitor of astrocyte and microglia in order to test its effect on the axonal growth-promoting function of aldynoglia precursor cells. We obtained an increase in GFAP expression corresponding to the expected glial phenotype for aldynoglia cells cultured in alkaline fibrin. In co-cultures of dorsal root ganglia (DRG) and aldynoglia, the axonal growth promotion of DRG neurons by aldynoglia was not affected. We observed that the neural precursor cells first clustered together and then formed niches from which aldynoglia cells grew and connected to groups of adjacent cells. We conclude that the combination of alkaline fibrin with synthetic sulfoglycolipid Tol-51 increased cell adhesion, cell migration, fasciculation, and axonal growth capacity, promoted by aldynoglia cells. There was no negative effect on the behavior of aldynoglia cells after the addition of sulfoglycolipid Tol-51, suggesting that a combination of aldynoglia plus alkaline fibrin and Tol-51 compound could be useful as a therapeutic strategy for tSCI repair.

Notch1 signaling pathway promotes growth and metastasis of gastric cancer via modulating CDH5.

To explore the underlying molecular mechanism of Notch1/cadherin 5 (CDH5) pathway in modulating in cell malignant behaviors of gastric cancer (GC). We performed bioinformatic analyses to screen the potential target genes of Notch1 from cadherins in GC. Western blot and RT-PCR were conducted to detect CDH5 expression in GC tissues and cells. We utilized chromatin immunoprecipitation (CHIP) assays to assess the interaction of Notch1 with CDH5 gene. The effects of Notch1/CDH5 axis on the proliferation, invasion, migration and vasculogenic mimicry in GC cells were evaluated by EdU, wound healing, transwell, and tubule formation assays. Significantly increased CDH5 expression was found in GC tissues compared with paracancerous tissues and associated to clinical stage and poor overall survival (OS) in patients with GC. Notch1 positively regulate the expression of CDH5 in GC cells. CHIP assays validated that CDH5 was a direct target of Notch1. In addition, Notch1 upregulation enhanced the proliferation, migration, invasion and vasculogenic mimicry capacity of GC cells, which could be attenuated by CDH5 silencing. These results indicated Notch1 upregulation enhanced GC malignant behaviors by triggering CDH5, suggesting that targeting Notch1/CDH5 axis could be a potential therapeutic strategy for GC progression.

METTL3 and IGF2BP1-Mediated m6A Modification of ZHX2 Promotes Tumor Property of Renal Cell Carcinoma

Introduction: Renal cell carcinoma (RCC) is a common type of kidney cancer with limited treatment options and a high mortality rate. Therefore, it is essential to understand the role and mechanism of key genes in RCC development and progression. This study aimed to analyze the role of zinc fingers and homeoboxes 2 (ZHX2) in RCC and the underlying mechanism. Methods: RNA expression was analyzed by quantitative real-time polymerase chain reaction, while protein expression was analyzed by Western blotting assay and immunohistochemistry assay. Cell viability was evaluated using CCK-8 assay, and cell proliferation was assessed by EdU assay. The rate of cell apoptosis was quantified by flow cytometry. Transwell assays were conducted to analyze cell migration and invasion. The sphere formation assay was performed to assess the formation of microspheres. Additionally, m6A RNA immunoprecipitation assay and RNA immunoprecipitation assay were utilized to investigate the relationship between ZHX2 and two proteins, methyltransferase like 3 (METTL3) and insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1). The stability of ZHX2 mRNA was analyzed through the Actinomycin D assay. Furthermore, a xenograft mouse model assay was conducted to analyze the effect of ZHX2 overexpression and METTL3 silencing on RCC cell tumor properties in vivo. Results: ZHX2 expression was upregulated in both RCC tissues and cells when compared with healthy renal tissues and human renal cortex proximal convoluted tubule epithelial cells. Depletion of ZHX2 inhibited RCC cell proliferation, migration, invasion, and spheroid-forming capacity but promoted cell apoptosis. Moreover, it was found that METTL3-mediated m6A methylation of ZHX2 and IGF2BP1 also stabilized ZHX2 through m6A methylation modification. Furthermore, ZHX2 overexpression showed a potential for attenuating the effects induced by METTL3 silencing and counteracted the inhibitory effect of METTL3 depletion on tumor formation in vivo. Conclusion: METTL3 and IGF2BP1-mediated m6A modification of ZHX2 promoted RCC progression. The finding suggests that ZHX2 may serve as a potential therapeutic target in RCC, providing valuable insights for future clinical interventions.

LAV-BPIFB4 and CXCR4 physically interact and potentiate the migration capacity of endothelial cells during hypoxic injury

LAV-BPIFB4 and CXCR4 physically interact and potentiate the migration capacity of endothelial cells during hypoxic injury

Effect of acacetin on inhibition of apoptosis in Helicobacter pylori-infected gastric epithelial cell line.

Helicobacter pylori (H. pylori) infection can cause extensive apoptosis of gastric epithelial cells, serving as a critical catalyst in the progression from chronic gastritis, gastrointestinal metaplasia, and atypical gastric hyperplasia to gastric carcinoma. Prompt eradication of H. pylori is paramount for ameliorating the pathophysiological conditions associated with chronic inflammation of the gastric mucosa and the primary prevention of gastric cancer. Acacetin, which has multifaceted pharmacological activities such as anti-cancer, anti-inflammatory, and antioxidative properties, has been extensively investigated across various domains. Nevertheless, the impact and underlying mechanisms of action of acacetin on H. pylori-infected gastric mucosal epithelial cells remain unclear. To explore the defensive effects of acacetin on apoptosis in H. pylori-infected GES-1 cells and to investigate the underlying mechanisms. GES-1 cells were treated with H. pylori and acacetin in vitro. Cell viability was assessed using the CCK-8 assay, cell mortality rate via lactate dehydrogenase assay, alterations in cell migration and healing capacities through the wound healing assay, rates of apoptosis via flow cytometry and TUNEL staining, and expression levels of apoptosis-associated proteins through western blot analysis. H. pylori infection led to decreased GES-1 cell viability, increased cell mortality, suppressed cell migration, increased rate of apoptosis, increased expressions of Bax and cle-caspase3, and decreased Bcl-2 expression. Conversely, acacetin treatment enhanced cell viability, mitigated apoptosis induced by H. pylori infection, and modulated the expression of apoptosis-regulatory proteins by upregulating Bcl-2 and downregulating Bax and cleaved caspase-3. Acacetin significantly improved GES-1 cell viability and inhibited apoptosis in H. pylori-infected GES-1 cells, thereby exerting a protective effect on gastric mucosal epithelial cells.

A chitosan-based light-curing hydrogel dressing for accelerated healing of infected wounds

Bacterial infections, excessive reactive oxygen species (ROS) accumulation and a persistent inflammatory response severely impede the wound healing process. In this study, we developed a novel multifunctional hydrogel dressing (LCPN) based on lipoic acid modified chitosan (LAMC), polypyrrole nanoparticles (PPy NPs) and nicotinamide mononucleotide (NMN) for accelerated healing of infected wounds. The synthesized LCPN hydrogel has several properties. Modification of lipoic acid significantly enhances the water solubility of chitosan making it easier to dissolve and absorb wound secretions. Interestingly, owing to the breaking and restructuring of disulfide bonds, LCPN hydrogel can be quickly bonded under UV light without relying on photoinitiators. In addition, the incorporation of PPy NPs not only enhances the electrical conductivity of LCPN hydrogel, but also confers photothermal antimicrobial capability to LCPN hydrogel. More importantly, the sustained release of NMN in LCPN hydrogel can significantly enhance cell proliferation, migration and antioxidant capacity, which is conducive to accelerated wound healing. In vitro and in vivo experiments have shown that LCPN hydrogel has excellent biocompatibility and the ability to promote wound healing. Therefore, the prepared multifunctional hydrogel is expected to be used as a novel dressing to accelerate wound healing.

Effect of ZCRB1 on the glycolysis via PKM2-HIF1α axis and promotion of immune tolerance in nasopharyngeal carcinoma.

130 Background: Nasopharyngeal carcinoma (NPC) is characterized by abundant lymphocyte infiltration. The failure of immune checkpoint inhibitors in NPC may be related to immune tolerance. Methods: Bioinformatics analysis was performed to identify the correlation between the ZCRB1 mRNA level and immune cell infiltration in NPC tissues. Quantitative real time PCR and immunoblotting were used to determine the mRNA and protein expression, respectively. Functional experiments (CCK8, colony formation assay, cell migration assay and tumor xenograft) were used to explore the effect of ZCRB1 on the malignant phenotype in NPC cells. Glucose and lactate measurement assays as well as Seahorse XF glycolysis stress assay, were conducted to investigate the effects of ZCRB1 on glycolysis in NPC cells. RNA sequencing, QPCR and minigene analysis were used to confirm the splicing switch between PKM1 and PKM2. Luciferase assay was performed to detect the transcription activity of HIF1-α. Finally, correlation analysis between the ZCRB1 expression and glycolysis level was performed to in NPC samples. Results: The expression of ZCRB1 was negatively correlated with checkpoint-related genes and immune regulation-related gene sets in head and neck cancer. Likewise, in NPC, the expression of ZCRB1 was negatively correlated with lymphocyte infiltration, tumor immune microenvironment score and tumor-infiltrating CD4+ T cells, CD8+ T cells, and dendritic cells. Low expression of ZCRB1 was associated with better prognosis. Functional assays demonstrated that ZCRB1 significantly promoted NPC cells growth, clonogenicity, migratory capacity and glycolysis. Mechanistically, ZCRB1 was mainly involved in the hypoxic signaling pathway. Cells with ZCRB1 downregulation downregulated glycolysis-related genes and significantly reduced the transcriptional activity of HIF-1α. Moreover, minigene assays showed that the intron 9 region spliced by PKM in cells with ZCRB1 downregulation. Finally, bioinformatics analysis showed that the mRNA expression of ZCRB1 was positively correlated with the immune infiltration score significantly during glycolysis. Conclusions: ZCRB1 promotes the growth and migration of nasopharyngeal carcinoma cells and is associated with immune tolerance. Further, ZCRB1 accelerates the glycolysis via enhancing the transcriptional activity of HIF-1α through increasing PKM2 isoform splicing, providing a scientific basis for tumor metabolic reprogramming to reshape the immune microenvironment.

Botulinum toxin type A inhibits the formation of hypertrophic scar through the JAK2/STAT3 pathway.

Hypertrophic scar (HS) is a fibrous proliferative disorder that occurs in the dermis after skin injury. Studies have confirmed that Botulinum toxin type A (BTA) is effective in scar prevention and treatment. However, the specific mechanism remains uncertain. Hypertrophic scar fibroblasts (HSFs) and normal skin fibroblasts (NSFs) from the skin tissues of HS patients were isolated and cultured. Western blot analysis was conducted to measure the expression of JAK2/STAT3 pathway-related proteins. HSFs were treated with the JAK2 inhibitor (AG490) or agonist (C-A1). The CCK-8 assay, EdU staining, scratch-wound assay and transwell assay were used to examine the biological properties of HSFs. Western blot, immunofluorescence, and Sirius red staining were used to assess the fibrosis of HSFs. Additionally, a mouse full-thickness wound model was constructed to investigate the role of BTA in wound healing. The results showed that the JAK2 and STAT3 phosphorylation levels were markedly increased in HS tissues and HSFs. AG490 treatment reduced cell viability, proliferation and migration capacity, and inhibited the fibrosis of HSFs, whereas C-A1 treatment had the opposite effect. BTA treatment inhibited the JAK2/STAT3 pathway. BTA reduced cell viability, proliferation and migration ability, and inhibited the fibrosis of HSFs, while C-A1 intervention weakened the impact of BTA. Meanwhile, BTA promoted wound healing and reduced collagen deposition in vivo. In conclusion, BTA inhibited the JAK2/STAT3 pathway, which in turn hindered the proliferation, migration and fibrosis of HSFs, and promoted wound healing in mice.

FASN promotes anoikis resistance in colorectal liver metastases through the ERK1/2 pathway

PurposeColorectal cancer (CRC) is recognized as the third most common form of malignancy, with the liver frequently serving as the main site for metastasis. Anoikis resistance (AR) is critical in colorectal cancer liver metastases (CRLM). Fatty acid synthase (FASN), essential in lipid synthesis, mediates AR in many cancers. The present research examines the function of FASN in ERK1/2-mediated AR in CRLM and evaluates its therapeutic potential. MethodsWe performed scratch and migration experiment to evaluate the migration capacity of the LoVo cells. Flow cytometry was employed to identify cell apoptosis. The levels of FASN, p-ERK1/2, and proteins related to apoptosis was analyzed by Western blot. The mRNA level of FASN was determined by q-PCR after FASN silencing. In addition, we used an intrasplenic liver metastasis model of nude to assess the effect of FASN on CRLM. ResultsIn vitro experiments showed that after FASN silencing, the cell apoptosis rate was increased, migration capability was notably decreased, the expression of p-ERK1/2, the proteins related to anti-apoptotic were significantly decreased, and the proteins related to apoptosis were significantly increased. In vivo experiments showed that AR significantly increased the number of liver metastatic foci, whereas FASN silencing significantly inhibited CRLM. ConclusionThese results suggest that FASN silencing suppressed AR through the ERK 1/2 pathway, which in turn suppressed CRLM.

CRISPR/Cas9-mediated suppression of A4GALT rescues endothelial cell dysfunction in a fabry disease vasculopathy model derived from human induced pluripotent stem cells

Background and aimsThe objective of this study was to investigate the efficacy of CRISPR/Cas9-mediated A4GALT suppression in rescuing endothelial dysfunction in Fabry disease (FD) endothelial cells (FD-ECs) derived from human induced pluripotent stem cells (hiPSCs). MethodsWe differentiated hiPSCs (WT (wild-type), WTC-11), GLA-mutant hiPSCs (GLA-KO, CMC-Fb-002), and CRISPR/Cas9-mediated A4GALT-KO hiPSCs (GLA/A4GALT-KO, Fb-002-A4GALT-KO) into ECs and compared FD phenotypes and endothelial dysfunction. We also analyzed the effect of A4GALT suppression on reactive oxygen species (ROS) formation and transcriptome profiles through RNA sequencing. ResultsGLA-mutant hiPSC-ECs (GLA-KO and CMC-Fb-002) showed downregulated expression of EC markers and significantly reduced α-GalA expression with increased Gb-3 deposition and intra-lysosomal inclusion bodies. However, CRISPR/Cas9-mediated A4GALT suppression in GLA/A4GALT-KO and Fb-002-A4GALT-KO hiPSC-ECs increased expression levels of EC markers and rescued these FD phenotypes. GLA-mutant hiPSC-ECs failed to form tube-like structure in tube formation assays, showing significantly decreased migration of cells into the scratched wound area. In contrast, A4GALT suppression improved tube formation and cell migration capacity. Western blot analysis revealed that MAPK and AKT phosphorylation levels were downregulated while SOD and catalase were upregulated in GLA-KO hiPSC-ECs. However, suppression of A4GALT restored these protein alterations. RNA sequencing analysis demonstrated significant transcriptome changes in GLA-mutant EC, especially in angiogenesis, cell death, and cellular response to oxidative stress. However, these were effectively restored in GLA/A4GALT-KO hiPSC-ECs. ConclusionsCRISPR/Cas9-mediated A4GALT suppression rescued FD phenotype and endothelial dysfunction in GLA-mutant hiPSC-ECs, presenting a potential therapeutic approach for FD-vasculopathy.

Single-cell sequencing combined with spatial transcriptomics reveals that the IRF7 gene in M1 macrophages inhibits the occurrence of pancreatic cancer by regulating lipid metabolism-related mechanisms.

The main focus of this study is to explore the molecular mechanism of IRF7 regulation on RPS18 transcription in M1-type macrophages in pancreatic adenocarcinoma (PAAD) tissue, as well as the transfer of RPS18 by IRF7 via exosomes to PAAD cells and the regulation of ILF3 expression. By utilising single-cell RNA sequencing (scRNA-seq) data and spatial transcriptomics (ST) data from the Gene Expression Omnibus database, we identified distinct cell types with significant expression differences in PAAD tissue. Among these cell types, we identified those closely associated with lipid metabolism. The differentially expressed genes within these cell types were analysed, and target genes relevant to prognosis were identified. Flow cytometry was employed to assess the expression levels of target genes in M1 and M2 macrophages. Cell lines with target gene knockout were constructed using CRISPR/Cas9 editing technology, and cell lines with target gene knockdown and overexpression were established using lentiviral vectors. Additionally, a co-culture model of exosomes derived from M1 macrophages with PAAD cells was developed. The impact of M1 macrophage-derived exosomes on the lipid metabolism of PAAD cells in the model was evaluated through metabolomics analysis. The effects of M1 macrophage-derived exosomes on the viability, proliferation, division, migration and apoptosis of PAAD cells were assessed using MTT assay, flow cytometry, EdU assay, wound healing assay, Transwell assay and TUNEL staining. Furthermore, a mouse PAAD orthotopic implantation model was established, and bioluminescence imaging was utilised to assess the influence of M1 macrophage-derived exosomes on the intratumoural formation capacity of PAAD cells, as well as measuring tumour weight and volume. The expression of proliferation-associated proteins in tumour tissues was examined using immunohistochemistry. Through combined analysis of scRNA-seq and ST technologies, we discovered a close association between M1 macrophages in PAAD samples and lipid metabolism signals, as well as a negative correlation between M1 macrophages and cancer cells. The construction of a prognostic risk score model identified RPS18 and IRF7 as two prognostically relevant genes in M1 macrophages, exhibiting negative and positive correlations, respectively. Mechanistically, it was found that IRF7 in M1 macrophages can inhibit the transcription of RPS18, reducing the transfer of RPS18 to PAAD cells via exosomes, consequently affecting the expression of ILF3 in PAAD cells. IRF7/RPS18 in M1 macrophages can also suppress lipid metabolism, cell viability, proliferation, migration, invasion and intratumoural formation capacity of PAAD cells, while promoting cell apoptosis. Overexpression of IRF7 in M1 macrophages may inhibit RPS18 transcription, reduce the transfer of RPS18 from M1 macrophage-derived exosomes to PAAD cells, thereby suppressing ILF3 expression in PAAD cells, inhibiting the lipid metabolism pathway, and curtailing the viability, proliferation, migration, invasion of PAAD cells, as well as enhancing cell apoptosis, ultimately inhibiting tumour formation in PAAD cells in vivo. Targeting IRF7/RPS18 in M1 macrophages could represent a promising immunotherapeutic approach for PAAD in the future.

METTL3/IGF2BP1 influences the development of non-small-cell lung cancer by mediating m6A methylation modification of TRPV1.

Methyltransferase 3 (METTL3) accelerates N6-methyladenosine (m6A) modifications and affects cancer progression, including non-small-cell lung cancer (NSCLC). In this study, we aimed to explore the regulatory mechanisms of METTL3 underling NSCLC. Immunohistochemical assay, quantitative real-time polymerase chain reaction (qRT-PCR)assay, and western blot assay were conducted for gene expression. MTT assay and colony formation assay were performed to explore cell proliferation capacity. Cell apoptosis and THP-1 cell polarization were estimated by flow cytometry analysis. Cell migration and invasion capacities were evaluated by transwell assay. Methylated RNA immunoprecipitation assay, dual-luciferase reporter assay, actinomycin D treatment and RIP assay were performed to analyze the relationships of METTL3, insulin-like growth factor 2 mRNA binding protein 1 (IGF2BP1), and transient receptor potential cation channel subfamily V member 1 (TRPV1). The functions of METTL3 and TRPV1 in vivo were investigated through establishing the murine xenograft model. TRPV1 expression was upregulated in NSCLC and related poor prognosis. TRPV1 silencing inhibited NSCLC cell growth and metastasis, induced NSCLC cell apoptosis, and repressed M2 macrophage polarization. The results showed that METTL3 and IGF2BP1 could regulate TRPV1 expression through m6A methylation modification. Moreover, METTL3 deficiency inhibited NSCLC cell growth, metastasis, and M2 macrophage polarization and facilitated NSCLC cell apoptosis, while TRPV1 overexpression restored the impacts. In addition, METTL3 knockdown restrained tumor growth in vivo via regulating TRPV1 expression. METTL3 bound to IGF2BP1 and enhanced IGF2BP1's m6A recognition of TRPV1 mRNA, thereby promoting NSCLC cell growth and metastasis, and inhibiting M2 macrophage polarization.

Calcium-sensing receptor regulates the angiogenic differentiation of LPS-treated human dental pulp cells via the phosphoinositide 3-kinase/Akt pathway invitro.

The purpose of this study was to investigate the role of calcium-sensing receptor (CaSR) in the angiogenic differentiation of lipopolysaccharide (LPS)-treated human dental pulp cells (hDPCs). The LPS-induced hDPCs were cultured in the medium with different combinations of CaSR agonist R568 and antagonist Calhex231. The cell proliferation, migration, and angiogenic capacity were measured by Cell Counting Kit-8 (CCK-8), scratch wound healing, and tube formation assays, respectively. Enzyme-linked immunosorbent assay (ELISA), quantitative real-time polymerase chain reaction (qRT-PCR), and western blot were conducted to determine the gene/protein expression of CaSR, inflammatory mediators, and angiogenic-associated markers. The activation of phosphoinositide 3-kinase (PI3K) and protein kinase B (Akt) was assessed by western blot analysis. The cell proliferation was elevated in response to R568 or Calhex231 exposure, but an enhanced cell migration was only found in cultures supplemented with Calhex231. Furthermore, R568 was found to potentiate the formation of vessel-like structure, up-regulated the protein expression of tumour necrosis factor (TNF)-α, vascular endothelial growth factor (VEGF), and stromal cell-derived factor (SDF)-1; comparable influences were also observed in R568-stimulated cells in the presence of PI3K inhibitor LY294002. In contrast, Calhex231 obviously inhibited the tube formation and VEGF protein level, whereas promoted the production of IL-6, TNF-α, and eNOS; however, in the presence of LY294002, Calhex231 showed a significant promotion on the protein expression of CaSR, VEGF, and SDF-1. In addition, R568 exhibited a promotive action on the Akt phosphorylation, which can be reversed by LY294002. Our results demonstrated that CaSR can regulate the angiogenic differentiation of LPS-treated hDPCs with an involvement of the PI3K/Akt signalling pathway.