Gene Expression In Cells Research Articles

Zi Shen Wan Fang repaired blood–brain barrier integrity in diabetic cognitive impairment mice via preventing cerebrovascular cells senescence

BackgroundBlood–brain barrier (BBB) integrity disruption is a key pathological link of diabetes-induced cognitive impairment (DCI), but the detailed mechanism of how the diabetic environment induces BBB integrity disruption is not fully understood. Our previous study found that Zi Shen Wan Fang (ZSWF), an optimized prescription consisting of Anemarrhenae Rhizoma (Anemarrhena asphodeloides Bge.), Phellodendri Chinensis Cortex (Phellodendron chinense Schneid.) and Cistanches Herba (Cistanche deserticola Y.C.Ma) has excellent efficacy in alleviating DCI, however, whether its mechanism is related to repairing BBB integrity remains unclear. This study aims to reveal the mechanism of BBB integrity destruction in DCI mice, and to elucidate the mechanism by which ZSWF repairs BBB integrity and improves cognitive function in DCI mice.MethodsDiabetic mouse model was established by feeding a 60% high-fat diet combined with a single intraperitoneal injection of 120 mg/kg streptozotocin (STZ). DCI mice were screened with morris water maze (MWM) after 8 weeks of sustained hyperglycemic stimulation. ZSWF was administered daily at doses of 9.36 and 18.72 g/kg for 8 weeks. Cognitive function was evaluated using MWM, blood–brain-barrier (BBB) integrity was tested using immunostaining and western blot, the underlying mechanisms were explored using single-cell RNA sequencing (scRNA-seq), validation experiments were performed with immunofluorescence analysis, and the potential active ingredients of ZSWF against cerebrovascular senescence were predicted using molecular docking. Moreover, cerebral microvascular endothelial cells were cultured, and the effects of mangiferin on the expression of p21 and Vcam1 were investigated by immunofluorescence staining and RT-qPCR.ResultsZSWF treatment significantly ameliorated cognitive function and repaired BBB integrity in DCI mice. Using scRNA-seq, we identified 14 brain cell types. In BBB constituent cells (endothelial cells and pericytes), we found that Cdkn1a and senescence-associated secretory phenotype (SASP) genes were significantly overexpressed in DCI mice, while ZSWF intervention significantly inhibited the expression of Cdkn1a and SASP genes in cerebrovascular cells of DCI mice. Moreover, we also found that the communication between brain endothelial cells and pericytes was decreased in DCI mice, while ZSWF significantly increased the communication between them, especially the expression of PDGFRβ in pericytes. Molecular docking results showed that mangiferin, the blood component of ZSWF, had a stronger affinity with the upstream proteins of p21. In vitro experiments showed that high glucose significantly increased the expression of p21 and Vcam1 in bEnd.3 cells, while mangiferin significantly inhibited the expression of p21 and Vcam1 induced by high glucose.ConclusionOur study reveals that ZSWF can ameliorate cognitive function in DCI mice by repairing BBB integrity, and the specific mechanism of which may be related to preventing cerebrovascular cells senescence, and mangiferin is its key active ingredient.Graphical

Polymer-Based Nanoparticles as Efficient Non-Viral Vectors for Gene Delivery in CAR-T Cell Therapy

Chimeric Antigen Receptor (CAR)-T cell therapy has emerged as a revolutionary method in cancer immunotherapy, achieving notable success in treating hematological malignancies. However, its effectiveness in solid tumors is limited, and the therapy faces challenges such as severe toxicities and high production costs. Polymer-based nanoparticles (PNPs) are gaining attention as a non-viral gene delivery system that can help address these issues, providing greater safety, scalability, and cost-effectiveness compared to viral vectors. This mini-review delves into various types of PNPs, including cationic, biodegradable, and stimuli-responsive polymers, showcasing their mechanisms for cellular uptake and sustained gene expression in CAR-T cells. Innovations like lipid-polymer hybrid nanoparticles and combination therapies improve gene delivery efficiency and therapeutic outcomes. Despite progress, challenges, including toxicity and immunogenicity, remain, prompting strategies such as surface modifications and targeting ligands. Recent advancements illustrate the potential of hybrid nanoparticles in enhancing CAR-T therapies, particularly for solid tumors, highlighting their critical role in cancer immunotherapy advancements.

Exploring Beta-sitosterol’s Role in Breast Cancer Treatment via Integrated Network Pharmacological Analysis and In Vitro Validation

Background Beta-sitosterol, a phytosterol similar to cholesterol, is found in various plants and is recognized for its potential anticancer properties; still, the mechanism of breast cancer (BC) remains elusive. Purpose This study investigates the potential targets of beta-sitosterol in BC using network pharmacological analysis and in vitro validation. Methods Targets of beta-sitosterol and BC were identified from online databases, including Swiss Target Prediction, SuperPred, GeneCards, and DisGeNET. Protein–protein interactions of common targets were analyzed using STRING version 11.0, and network construction and core target screening were performed with Cytoscape 3.8.0. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis and gene ontology (GO) annotation were conducted using the ShinyGO web tool. Molecular docking analysis was done with AutoDock Vina and the in vitro validation, including cell viability, apoptosis, and gene expression analysis was done using MTT assay, acridine orange/ethidium bromide staining, and polymerase chain reaction (PCR), respectively. Results Sixty-eight common targets for beta-sitosterol and BC were identified from 209 beta-sitosterol targets and 1,350 BC-related genes. Five core targets (HIF1A, ESR1, STAT3, TNF, and MAPK3) were identified. GO analysis linked common targets to biological processes, cellular components, and molecular functions. KEGG pathway analysis showed enrichment in pathways such as central carbon metabolism in cancer, vascular endothelial growth factor signaling, and prolactin signaling. Beta-sitosterol’s impact on MCF7 cell viability was assessed via MTT assay, and its molecular effects were explored through PCR. Results demonstrated beta-sitosterol’s ability to hinder tumor progression by modulating genes involved in ESR1, mitogen-activated protein kinase, and tumor necrosis factor signaling pathways. Conclusion These findings highlight beta-sitosterol’s potential as an anticancer agent, offering new perspectives for clinical investigations through the modulation of key signaling pathways in BC.

Interplay between Netrin-1 and Norrin controls arteriovenous zonation of blood–retina barrier integrity

The integrity of the blood-retina barrier (BRB) is crucial for phototransduction and vision, by tightly restricting transport of molecules between the blood and surrounding neuronal cells. Breakdown of the BRB leads to the development of retinal diseases. Here, we show that Netrin-1/Unc5b and Norrin/Lrp5 signaling establish a zonated endothelial cell gene expression program that controls BRB integrity. Using single-cell RNA sequencing (scRNA-seq) of postnatal BRB-competent mouse retina endothelial cells (ECs), we identify >100 BRB genes encoding Wnt signaling components, tight junction proteins, and ion and nutrient transporters. We find that BRB gene expression is zonated across arteries, capillaries, and veins and regulated by opposing gradients of the Netrin-1 receptor Unc5b and Lrp5-β-catenin signaling between retinal arterioles and venules. Mice deficient for Ntn1 or Unc5b display more BRB leakage at the arterial end of the vasculature, while Lrp5 loss of function causes predominantly venular BRB leakage. ScRNA-seq of Ntn1 and Unc5b mutant ECs reveals down-regulated β-catenin signaling and BRB gene expression that is rescued by Ctnnb1 overactivation, along with BRB integrity. Mechanistically, we demonstrate that Netrin-1 and Norrin additively enhance β-catenin transcriptional activity and Lrp5 phosphorylation via the Discs large homologue 1 (Dlg1) scaffolding protein, and endothelial Lrp5-Unc5b function converges in protection of capillary BRB integrity. These findings explain how arteriovenous zonation is established and maintained in the BRB and reveal that BRB gene expression is regulated at the level of endothelial subtypes.

Distinct Tissue-Dependent Composition and Gene Expression of Human Fetal Innate Lymphoid Cells.

The human fetal immune system starts to develop in the first trimester and likely plays a crucial role in fetal development and maternal-fetal tolerance. Innate lymphoid cells (ILCs) are the earliest lymphoid cells to arise in the human fetus. ILCs consist of natural killer (NK) cells, ILC1s, ILC2s, and ILC3s that all share a common lymphoid origin. Here, we studied fetal ILC subsets, mainly NK cells and ILC3s and their potential progenitors, across human fetal tissues. Our results show that fetal ILC subsets have distinct distribution, developmental kinetics, and gene expression profiles across human fetal tissues. Furthermore, we identify CD34+RORγt+Eomes- and CD34+RORγt+Eomes+ cells in the fetal intestine, indicating that tissue-specific ILC progenitors exist already during fetal development.

In silico, invitro, and exvivo analysis reveals miR-27a-3p and miR-155-5p as key microRNAs for glioblastoma progression: Insights into Th1 differentiation and apoptosis induction.

We explored key microRNAs (miRNAs) related to tumorigenesis and immune modulation in glioblastoma (GBM), employing in silico, invitro, and exvivo analysis along with an assessment of the cellular impacts resulting from miRNA inhibition. GBM and T cells miRNA expression profiles from public datasets were used to evaluate differentially expressed miRNAs (DEmiRNAs). Some DEmiRNAs were chosen for validation in GBM cell lines, primary cell cultures, and brain tumor patient samples, using RT-qPCR. Target genes and pathways were identified with bioinformatic analyses. In silico functional enrichment analysis revealed that miR-27a-3p and miR-155-5p modulate immune, metabolic, and GBM-related pathways. A172 cells were transfected with miRNA inhibitors and the effects on cellular processes and immunomodulation were analyzed by co-culture assays and flow cytometry. Upon validation, miR-27a-3p and miR-155-5p miRNAs expressions were consistently increased. Inhibiting these two miRNAs reduced cell viability, but only the inhibition of miR-27a-3p led to apoptosis. Co-culture assays showed an increase in Th1 cells along with elevated Th1/Treg and Th17/Treg ratios, and an increase in Th17 cells exclusively with miR-155-5p inhibition. Immune cells' gene expression modulation induced an antitumor profile, concomitant with an increase in the expression of apoptotic genes in cancer cells after co-culture. This study unveils potential targets for immune and tumor regulation, highlighting overexpressed miRNAs modulation as a novel therapeutic approach for GBM.

Integrated Analysis of Gene Expression and Immune Cell Infiltration Reveals Dysregulated Genes and miRNAs in Acute Kidney Injury.

Acute Kidney Injury (AKI) is a multifaceted condition characterised by rapid deterioration of renal function, often precipitated by diverse etiologies. A comprehensive understanding of the molecular underpinnings of AKI is pivotal for identifying potential diagnostic markers and therapeutic targets. This study utilised bioinformatics to elucidate gene expression and immune infiltration in AKI. Publicly available mRNA and miRNA datasets were harnessed to discern differentially expressed genes (DEGs) and miRNAs in AKI. The CIBERSORT algorithm was employed to quantify immune cell infiltration in AKI samples. Functional enrichment analyses were conducted to unravel the implicated biological processes. Furthermore, the expression of identified genes and miRNAs was validated by quantitative real-time PCR in an AKI model. Our study revealed significant dysregulation of three genes (Aspn, Clec2h, Tmigd1) and two miRNAs (mmu-miR-21a-3p, mmu-miR-223-3p) in AKI, each with p < 0.0001. These molecular markers are implicated in immune responses, tissue remodelling, and inflammation. We observed notable disturbances in specific immune cells, including activated and immature dendritic cells, M1 macrophages, and subsets of T cells (Treg, Th1, Th17). These alterations correlated significantly with AKI pathology, with dendritic cells and M1 macrophages showing p < 0.01, and T cell subsets demonstrating p < 0.05. These results highlight the intricate involvement of the immune system in AKI and indicate significant enrichment of pathways related to immune response, inflammation, and tissue remodelling, pointing to their pivotal roles in AKI pathophysiology. Our study underscored the significance of immune cell infiltration and dysregulated gene and miRNA expression in AKI. The identified genes (Clec2h, Aspn, and Tmigd1) and miRNAs (mmu-miR-21a-3p and mmu-miR-223-3p) offer potential diagnostic markers and therapeutic avenues for AKI. Subsequent investigations targeting these genes and miRNAs, along with the elucidated pathways, may augment the clinical management and outcomes for AKI patients.

Exploration of the Regulatory Network of Programmed Cell Death Genes in Rheumatoid Arthritis Based on Blood-Derived circRNA Transcriptome Information and Single-Cell Multi-omics Data.

Programmed cell death (PCD) and circular RNA (circRNA) have been found to involve in the pathogenesis of rheumatoid arthritis (RA). The aim of this study was to explore PCD mechanisms and gene regulatory networks in RA. RA related to circRNA, mRNA and single-cell data sets were obtained from the GEO database. The limma package was used to screen differentially expressed circRNA and differentially expressed genes (DEGs) of RA. The PCD gene set from literature was intersected with the DEGs of RA to obtain PCD-related DEGs of RA. The ENCORI database was used to predict and construct a competing endogenous RNAs (ceRNA) regulatory network to obtain key circRNAs and PCD-related DEGs. Hub genes were identified from the key PCD-related DEGs in the ceRNA regulatory network through LASSO regression, and a diagnostic model was constructed based on these hub genes. The expression of hub genes in various cells and stages was analyzed using single-cell datasets. Finally, the expression of key circRNAs and hub genes in peripheral blood of RA patients and healthy individuals was verified by PCR. In this study, a total of 71 differential circRNAs and 221 DEGs in RA were obtained, and 23 PCD-related DEGs were identified. Through ceRNA regulatory network, three key circRNAs (hsa_circ_0001241, hsa_circ_0089761, and hsa_circ_0001654) and four hub PCD-related DEGs. Among them, TXN and RRAGD were highly expressed, and PARP1 and TXNIP were lowly expressed in RA. Single-cell analysis revealed that these genes were significantly differentially expressed in myeloid cell subpopulation. PCR results indicated that among the 7 key factors, the expression of hsa_circ_0001241, hsa_circ_0089761, TXN, and RRAGD in RA was consistent with the results of bioinformatics analysis. Hsa_circ_0001241, hsa_circ_0089761, TXN and RRAGD may be potential biomarkers for RA, and their interactions may have significant implications for the pathology of RA.

Bioinformatic Analysis of Actin-Binding Proteins in the Nucleolus During Heat Shock

Background/Objectives: Actin plays a crucial role not only in the cytoplasm, but also in the nucleus, influencing various cellular behaviors, including cell migration and gene expression. Recent studies reveal that nuclear actin dynamics is altered by cellular stresses, such as DNA damage; however, the effect of heat shock on nuclear actin dynamics, particularly in the nucleolus, remains unclear. This study aims to elucidate the contribution of nucleolar actin to cellular responses under heat shock conditions. Methods: Nuclear actin dynamics in response to heat shock were investigated using nAC-GFP, a GFP-tagged actin chromobody, to visualize nuclear actin in HeLa cells. Bioinformatic analyses were also performed. Results: Heat shock induced the reversible assembly of nAC-GFP in the nucleolus, with disassembly occurring upon recovery in a heat shock protein (Hsp) 70-dependent manner. Because the nucleolus, formed via liquid–liquid phase separation (LLPS), sequesters misfolded proteins under heat shock to prevent irreversible aggregation, we hypothesized that nucleolar actin-binding proteins might also be sequestered in a similar manner. Using several databases, we identified 47 actin-binding proteins localized in the nucleolus and determined the proportion of intrinsically disordered regions (IDRs) known to promote LLPS. Our analysis revealed that many of these 47 proteins exhibited high levels of IDRs. Conclusions: The findings from our bioinformatics analysis and further cellular studies may help elucidate new roles for actin in the heat shock response.

Cell-Specific Control of Mammalian Gene Expression Using DNA Repair Inducible Ribozyme Switches.

The ability to control gene expression is vital for elucidating gene functions and developing next-generation therapeutics. Current techniques are challenged by the lack of cell-specific control designs or immunogenicity risk from foreign proteins. We develop a DNA repair inducible ribozyme switch that enables cell-specific control of gene expression in cells and in vivo. This strategy designs plasmids with a DNA lesion (8-oxoG and O6-MeG) site-specifically installed within the ribozyme encoding region, generating active hammerhead ribozyme for mRNA degradation due to transcriptional mutagenesis, whereas DNA repair yields a single-base mismatch in the ribozyme to abrogate its activity. This strategy is demonstrated to allow specific control of gene expression in cancer cells with overexpressed DNA repair enzymes such as MutY DNA glycosylase and O6-methylguanine-DNA-methyltransferases. It also shows the capability of conditionally regulating the expression of different proteins for signal reporting and gene editing, enabling DNA repair monitoring and targeted gene therapy in cancer cells. This strategy is demonstrated using the inducible CRISPR/Cas9 system for in vivo editing of oncogenic Polo-like kinase 1 in a mouse model, resulting in significant tumor growth suppression. The DNA repair inducible ribozyme switch may provide a compact system for cell-specific gene expression control toward precise gene therapy.

Cell‐Specific Control of Mammalian Gene Expression Using DNA Repair Inducible Ribozyme Switches

The ability to control gene expression is vital for elucidating gene functions and developing next‐generation therapeutics. Current techniques are challenged by the lack of cell‐specific control designs or immunogenicity risk from foreign proteins. We develop a DNA repair inducible ribozyme switch that enables cell‐specific control of gene expression in cells and in vivo. This strategy designs plasmids with a DNA lesion (8‐oxoG and O6‐MeG) site‐specifically installed within the ribozyme encoding region, generating active hammerhead ribozyme for mRNA degradation due to transcriptional mutagenesis, whereas DNA repair yields a single‐base mismatch in the ribozyme to abrogate its activity. This strategy is demonstrated to allow specific control of gene expression in cancer cells with overexpressed DNA repair enzymes such as MutY DNA glycosylase and O6‐methylguanine‐DNA‐methyltransferases. It also shows the capability of conditionally regulating the expression of different proteins for signal reporting and gene editing, enabling DNA repair monitoring and targeted gene therapy in cancer cells. This strategy is demonstrated using the inducible CRISPR/Cas9 system for in vivo editing of oncogenic Polo‐like kinase 1 in a mouse model, resulting in significant tumor growth suppression. The DNA repair inducible ribozyme switch may provide a compact system for cell‐specific gene expression control toward precise gene therapy.

Upregulation of HPV16E1 and E7 expression and FOXO3a mRNA downregulation in high-grade cervical neoplasia.

Cervical cancer remains a significant global health concern, ranking as the fourth most prevalent cancer among women worldwide. Human papillomaviruses (HPV) transcribe many genes that might be responsible for cervical cancer development. This study aims to investigate the correlation between the expression of HPV16 early genes and the mRNA expression of human FOXO3a, a tumor suppressor gene, in association with various stages of cervical precancerous lesions. Eighty-five positive HPV16 DNA cervical swab samples were recruited and categorized based on cytology stages, i.e., negative for intraepithelial lesion or malignancy (NILM), atypical squamous cells of undetermined significance (ASC-US), low-grade squamous intraepithelial lesion (LSIL), atypical squamous cell cannot exclude HSIL (ASC-H), high-grade squamous intraepithelial lesion (HSIL). RT-qPCR was performed to amplify HPV16E1, E4, E6, E6*I, E7, and human FOXO3a mRNA expression in all samples. The relative expression of those genes was calculated using GAPDH as a control. Detection of FOXO3a mRNA expression in the cervical cancer cell line by RT-qPCR and meta-analysis of FOXO3a expression using the RNA-Seq dataset by GEPIA2 were analyzed to support the conclusions. Among the cervical samples, HPV16E1 and E7 were significantly increased expression correlating to disease severity. HPV16E4 mRNA expression was 100% detected in all LSIL samples, with a significant increase observed from normal to LSIL stages. Conversely, FOXO3a mRNA expression decreased with disease severity, and the lowest expression was observed in HSIL/squamous cell carcinoma (SCC) samples. In addition, similar results of FOXO3a downregulation were also found in the cervical cancer cell line and RNA-Seq dataset of cervical cancer samples. HPV16 early mRNA levels, including E1 and E7, increase during cancer progression, and downregulation of FOXO3a mRNA is a characteristic of cervical cancer cells and HSIL/SCC. Additionally, HPV16E4 mRNA expression was consistently detected in all LSIL samples, suggesting the presence of active viral replication. These findings might lead to further investigation into the interplay between HPV gene expression and host cell factors for targeted therapeutic strategies in cervical cancer management.

Differential Expression of Neurodegeneration-Related Genes in SH-SY5Y Neuroblastoma Cells Under the Influence of Cyclophilin A: Could the Enzyme be a Likely Trigger and Therapeutic Target for Alzheimer's Disease?

The function and mechanism of Cyclophilin A (CypA) in modulating gene expression associated with Alzheimer's disease (AD) remain unclear. This multifunctional protein is found to be elevated in the cerebrospinal fluid (CSF) of individuals at risk for AD. The cytotoxic effects of CypA, including both wild-type and the mutant R55A, were assessed using the MTT assay. Prior to this evaluation, the purified recombinant protein was validated through enzymatic activity assays and western blot analysis. Following treatment with CypA and transient transfection using the CypA construct, real-time PCR (qRT-PCR) and western blotting were conducted to analyze the expression of factors involved in various signaling pathways, with an emphasis on inflammation, cell death, and intercellular communication. The findings indicate that CypA has a significant impact on the gene expression of factors associated with inflammation and the progression of AD in SH-SY5Y cells. It can be concluded that CypA is capable of regulating gene expression in SH-SY5Y cells, either in a manner dependent on or independent of its enzymatic activity. Additionally, the influence of this multifunctional protein on gene expression is contingent upon the specific site of action, as well as the dosage and duration of exposure to the cells.

Effects of Hypomethylating Agents on Gene Modulation in the Leukemic Microenvironment and Disease Trajectory in a Mouse Model of AML.

Hypomethylating agents (HMAs), such as decitabine and 5-azacytidine (AZA), are valuable treatment options for patients with acute myeloid leukemia that are ineligible for intensive chemotherapy. Despite providing significant extensions in survival when used alone or in combination, eventual relapse and resistance to HMAs are observed. The mechanisms leading to these outcomes are still not well defined and may, in part, be due to a focus on leukemic populations with limited information on the effects of HMAs on non-leukemic cells in the blood and other tissue compartments. In this study, we elucidated effects on immune-related gene expression in non-leukemic blood cells and the spleen during AZA treatment in leukemia-challenged mice. We observed significant changes in pathways regulating adhesion, thrombosis, and angiogenesis as well as a dichotomy in extramedullary disease sites that manifests during relapse. We also identify several genes that may contribute to the anti-leukemic activity of AZA in blood and spleen. Overall, this work has identified novel gene targets and pathways that could be further modulated to augment efficacy of HMA treatment.

Integrative multi-omics approach using random forest and artificial neural network models for early diagnosis and immune infiltration characterization in ischemic stroke.

Ischemic stroke (IS) is a significant global health issue, causing high rates of morbidity, mortality, and disability. Since conventional Diagnosis methods for IS have several shortcomings. It is critical to create new Diagnosis models in order to enhance existing Diagnosis approaches. We utilized gene expression data from the Gene Expression Omnibus (GEO) databases GSE16561 and GSE22255 to identify differentially expressed genes (DEGs) associated with IS. DEGs analysis using the Limma package, as well as GO and KEGG enrichment analyses, were performed. Furthermore, PPI networks were constructed using DEGs from the String database, and Random Forest models were utilized to screen key DEGs. Additionally, an artificial neural network model was developed for IS classification. Use the GSE58294 dataset to evaluate the effectiveness of the scoring model on healthy controls and ischemic stroke samples. The effectiveness of the scoring model was evaluated through AUC analysis, and CIBERSORT analysis was conducted to estimate the immune landscape and explore the correlation between gene expression and immune cell infiltration. A total of 26 significant DEGs associated with IS were identified. Metascape analysis revealed enriched biological processes and pathways related to IS. 10 key DEGs (ARG1, DUSP1, F13A1, NFIL3, CCR7, ADM, PTGS2, ID3, FAIM3, HLA-DQB1) were selected using Random Forest and artificial neural network models. The area under the ROC curve (AUC) for the IS classification model was found to be near 1, indicating its high accuracy. Additionally, the analysis of the immune landscape demonstrated elevated immune-related networks in IS patients compared to healthy controls. The study uncovers the involvement of specific genes and immune cells in the pathogenesis of IS, suggesting their importance in understanding and potentially targeting the disease.

Effect of Liquid Blood Concentrates on Cell Proliferation and Cell Cycle- and Apoptosis-Related Gene Expressions in Nonmelanoma Skin Cancer Cells: A Comparative In Vitro Study.

Nonmelanoma skin cancer (NMSC) presents a significant challenge to global healthcare due to its rising incidence, prompting the search for innovative treatments to overcome the limitations of current therapies. Our study aims to explore the potential effects of the liquid blood concentrate platelet-rich fibrin (PRF) on basal cell carcinoma cells (BCCs) and squamous cell carcinoma cells (SCCs) in order to obtain results that may lead to new possible adjunctive therapies for managing localized skin cancers, particularly NMSC. Basal cell carcinoma (BCC) cells and squamous cell carcinoma (SCC) cells were indirectly treated with PRF generated via different relative centrifugation forces, namely high and low RCF PRF, for 7 days. PRF-treated cells were comparatively analyzed for cell viability, proliferation and cell cycle- and apoptosis-related gene expression. Analysis of MTS assay results revealed a significant decrease in cell viability in both BCC and SCC cells following PRF treatment for 7 days. Ki-67 staining showed a decreased percentage of Ki-67-positive cells in both BCC and SCC cells after 2 days of treatment compared to the control group. The downregulation of CCND1 gene expression in both cell types at 2 days along with the upregulation of p21 and p53 gene expression in SCC cells demonstrated the effect of PRF in inhibiting cell proliferation and inducing cell cycle arrest, especially during the initial phases of treatment. Increased expression of caspase-8 and caspase-9 was observed, indicating the activation of both extrinsic and intrinsic apoptotic pathways by PRF treatment. Although the exact immunomodulatory properties of PRF require further investigation, the results of our basic in vitro studies are promising and might provide a basis for future investigations of PRF as an adjunctive therapy for managing localized skin cancers, particularly NMSC.

Targeting cuproptosis with nano material: new way to enhancing the efficacy of immunotherapy in colorectal cancer.

Colorectal cancer has emerged as one of the predominant malignant tumors globally. Immunotherapy, as a novel therapeutic methodology, has opened up new possibilities for colorectal cancer patients. However, its actual clinical efficacy requires further enhancement. Copper, as an exceptionally crucial trace element, can influence various signaling pathways, gene expression, and biological metabolic processes in cells, thus playing a critical role in the pathogenesis of colorectal cancer. Recent studies have revealed that cuproptosis, a novel mode of cell death, holds promise to become a potential target to overcome resistance to colorectal cancer immunotherapy. This shows substantial potential in the combination treatment of colorectal cancer. Conveying copper into tumor cells via a nano-drug delivery system to induce cuproptosis of colorectal cancer cells could offer a potential strategy for eliminating drug-resistant colorectal cancer cells and vastly improving the efficacy of immunotherapy while ultimately destroy colorectal tumors. Moreover, combining the cuproptosis induction strategy with other anti-tumor approaches such as photothermal therapy, photodynamic therapy, and chemodynamic therapy could further enhance its therapeutic effect. This review aims to illuminate the practical significance of cuproptosis and cuproptosis-inducing nano-drugs in colorectal cancer immunotherapy, and scrutinize the current challenges and limitations of this methodology, thereby providing innovative thoughts and references for the advancement of cuproptosis-based colorectal cancer immunotherapy strategies.

Effect of cigarette smoke on the proliferation, viability, gene expression, and cellular functions of adipose-derived mesenchymal stem cells from smoking and non-smoking donors.

Cigarette smoking negatively impacts mesenchymal stem cell functionality, including proliferation, viability, and differentiation potential. Adipose-derived mesenchymal stem cells (ADMSCs) are increasingly used for therapeutic purposes, but the specific effects of smoking in vivo on these cells are poorly understood. This study investigates the effects of cigarette smoke on the proliferation, viability, gene expression, and cellular functions of ADMSCs from smoking and non-smoking donors. In this study, ADMSCs were isolated from healthy smokers and non-smokers, and cell proliferation was assessed using the MTT assay, viability with apoptosis assays, mitochondrial membrane potential (MMP), and gene expression related to oxidative stress and cellular functions. Cell cycle analysis was also conducted. Our findings reveal a significant decrease in the proliferation of ADMSCs from smokers. Apoptosis assays showed reduced viable cells in smokers without a significant change in MMP, suggesting alternative pathways contributing to decreased viability. Gene expression analysis indicated the upregulation of genes associated with oxidative stress response and cellular defense mechanisms and the downregulation of genes related to inflammatory signaling, detoxification, and cellular metabolism. Cell cycle analysis indicates cycle arrest or delay in smokers, possibly due to stress and potential DNA damage. Smoking negatively affects ADMSCs' proliferation, viability, and function through oxidative stress and gene expression alterations. These findings highlight the importance of considering smoking status in ADMSC therapies and the need for further research to mitigate the effect of smoking on stem cells.

Extracellular vesicles and micro-RNAs in liver disease.

Progression of liver disease is dependent on intercellular signaling, including those mediated by extracellular vesicles (EVs). Within these EVs, microRNAs (miRNAs) are packaged to selectively silence gene expression in recipient cells for upregulating or downregulating a specific pathway. Injured hepatocytes secrete EV-associated miRNAs which can be taken up by liver sinusoidal endothelial cells (LSECs), immune cells, hepatic stellate cells (HSCs), and other cell types. In addition, these recipient cells will secrete their own EV-associated miRNAs to propagate a response throughout the tissue and the circulation. In this review, we comment on the implications of EV-miRNAs in the progression of alcohol-associated liver disease (ALD), metabolic dysfunction-associated steatohepatitis (MASH), viral and parasitic infections, liver fibrosis, and liver malignancies. We summarize how circulating miRNAs can be used as biomarkers and the potential of utilizing EVs and miRNAs as therapeutic methods to treat liver disease.

Spatial immune heterogeneity in a mouse tumor model after immunotherapy.

Cancer immunotherapy is increasingly used in clinical practice, but its success rate is reduced by tumor escape from the immune system. This may be due to the genetic instability of tumor cells, which allows them to adapt to the immune response and leads to intratumoral immune heterogeneity. The study investigated spatial immune heterogeneity in the tumor microenvironment and its possible drivers in a mouse model of tumors induced by human papillomaviruses (HPV) following immunotherapy. Gene expression was determined by RNA sequencing and mutations by whole exome sequencing. A comparison of different tumor areas revealed heterogeneity in immune cell infiltration, gene expression, and mutation composition. While the mean numbers of mutations with every impact on gene expression or protein function were comparable in treated and control tumors, mutations with high or moderate impact were increased after immunotherapy. The genes mutated in treated tumors were significantly enriched in genes associated with ECM metabolism, degradation, and interactions, HPV infection and carcinogenesis, and immune processes such as antigen processing and presentation, Toll-like receptor signaling, and cytokine production. Gene expression analysis of DNA damage and repair factors revealed that immunotherapy upregulated Apobec1 and Apobec3 genes and downregulated genes related to homologous recombination and translesion synthesis. In conclusion, this study describes the intratumoral immune heterogeneity, that could lead to tumor immune escape, and suggests the potential mechanisms involved.