Quantitative Real-time PCR Research Articles

Exploring the mechanism of berberine treatment for atherosclerosis combined with non-alcoholic fatty liver disease based on bioinformatic and experimental study

Atherosclerosis (AS) and Non-alcoholic fatty liver disease (NAFLD) are chronic metabolic disorders with high prevalence and significant health impacts. Both conditions share common pathophysiological pathways including abnormal lipid metabolism and inflammation. Berberine (BBR), an isoquinoline alkaloid, is known for its beneficial effects on various metabolic and cardiovascular disorders. This study investigates BBR’s impact on AS and NAFLD through bioinformatics analysis and experimental models. This study utilized various bioinformatics methods, including transcriptome analysis, weighted gene co-expression network analysis (WGCNA), machine learning, and molecular docking, to identify key genes and pathways involved in AS and NAFLD. Subsequently an animal model of AS combined with NAFLD was established using ApoE-/- mice fed a high-fat diet. The efficacy and mechanism of action of BBR were verified using methods such as hematoxylin and eosin (HE) staining, Oil Red O staining, and real-time quantitative PCR (RTqPCR). Through transcriptome analysis, WGCNA, and machine learning, this study identified 48 key genes involved in both AS and NAFLD. Function analysis revealed that the implicated genes were significantly involved in pathways like cytokine-cytokine receptor interaction, chemokine signaling, and IL-17 signaling pathway, suggesting their role in inflammation and immune responses. Single cell validation identified six key genes: dual specificity phosphatase 6 (DUSP6), chemokine ligand 3 (CCL3), complement component 5a receptor 1 (C5AR1), formyl peptide receptor 1 (FPR1), myeloid nuclear differentiation antigen (MNDA), and proviral integration site of murine 2(PIM2). Finally, molecular docking and animal experiments showed that BBR significantly reduced lipid deposits and inflammatory markers in liver and aortic tissues. In conclusion, BBR can improve AS combined with NAFLD by regulating genes like MNDA, PIM2, DUSP6, CCL3, C5AR1, and FPR1, with the mechanism related to inflammation control. The findings suggest potential clinical benefits of BBR in reducing the progression of both AS and NAFLD, warranting further investigation.

Exploring microRNA signatures in pediatric non-infectious uveitis: meta-analysis and molecular profiling of patient samples.

To find a distinct non-coding RNA characteristic for idiopathic uveitis in the pediatric population. To explore the autoimmune-related miRNA expression profile in pediatric patients with idiopathic uveitis (IU) and juvenile idiopathic arthritis-associated uveitis (JIA-AU) and find a common molecular background for idiopathic uveitis and other autoimmune diseases. The expression levels of miRNAs were analyzed by quantitative real-time PCR using serum samples from patients with idiopathic uveitis (n = 8), juvenile idiopathic arthritis-associated uveitis (n = 7), and healthy controls. We selected the most promising miRNAs from the original research papers: miR-16-5p, miR-26a-5p, miR-145-5p, and miR-451a as markers for juvenile idiopathic arthritis; miR-23a-3p, miR-29a-3p, miR-140-5p, miR-193a-5p, and miR-491-5p for uveitis in the adult population; and miR-125a-5p, miR-146a-5p, miR-155-5p, miR-223-5p, and miR-223-3p characteristic for both diseases and confirm their expression changes in serum from children with idiopathic uveitis. We comprehensively reviewed the literature enrolling the papers that met the inclusion criteria (miRNA and non-infectious uveitis/juvenile idiopathic arthritis) and performed target prediction analysis of appoint miRNAs. It additionally confirmed that altered miRNAs target the immunologically involved genes. Immunological-involved miRNAs such as miR-146a-5p and miR-155-5p show diverse expression levels in different patients as they interact with multiple targets. miR-204-5p is downregulated in both patient groups compared to healthy controls. miR-204-5p and miR-155-5p are candidates for molecular markers of autoimmune uveitis. We did not identify the miRNAs specific only to idiopathic uveitis, but for the first time in the pediatric population, we confirmed that this disease entity shares a molecular basis with other autoimmune diseases. Further studies are required to elucidate the molecular interactions among miRNAs, cytokines, and transcription factors within the intricate immune response, particularly in the eye.

CMTM6 promotes hepatocellular carcinoma invasion and metastasis and tumor-associated neutrophil immunoinfiltration through the Wnt/β-catenin pathway

BackgroundCMTM6 has been closely associated with the onset and progression of various tumor types. However, the precise mechanism by which CMTM6 operates in hepatocellular carcinoma remains elusive, necessitating further investigation.MethodsExpression levels of CMTM6 in hepatocellular carcinoma tissues and cells were analyzed using immunohistochemistry and quantitative real-time PCR. The correlation between CMTM6 expression in hepatocellular carcinoma tissues and clinical pathological characteristics, as well as patient prognosis, was investigated. Proliferation and apoptosis of hepatocellular carcinoma cells with silenced or overexpressed CMTM6 were assessed, alongside measurements of β-catenin and Wnt1 protein expression levels. In vivo research was conducted utilizing a murine subcutaneous transplantation model. Gene Set Enrichment Analysis (GSEA) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis were performed to elucidate the regulatory mechanism of CMTM6. Additionally, CD66b expression levels in tumor tissue were examined using immunohistochemistry, and the immune infiltration of CMTM6 and tumor-associated neutrophils (TANs) was analyzed.ResultsElevated expression levels of CMTM6 in hepatocellular carcinoma tissues and cells were found to be associated with poor patient prognosis. Overexpression of CMTM6 in hepatocellular carcinoma cells was demonstrated to promote cellular proliferation and inhibit apoptosis. Mechanistically, CMTM6 expression levels in hepatocellular carcinoma tissues were observed to positively correlate with β-catenin expression. GSEA and KEGG analysis revealed significant enrichment of CMTM6 in the Wnt/β-catenin pathway, indicating its involvement in pathway regulation. Furthermore, CMTM6 was found to be associated with immune infiltration of TANs in hepatocellular carcinoma tissues.ConclusionCMTM6 plays a pivotal role in the development and progression of hepatocellular carcinoma through regulation of the Wnt/β-catenin pathway via β-catenin. Moreover, CMTM6 demonstrates the capacity to promote immune infiltration of TANs in hepatocellular carcinoma tissues. Consequently, CMTM6 exhibits potential as both an early diagnostic marker and a novel therapeutic target for patients with hepatocellular carcinoma.

Construction of a prognostic model based on disulfidptosis-related genes and identification of CCNA2 as a novel biomarker for hepatocellular carcinoma

BackgroundDisulfidptosis, identified as an innovative form of cellular death subsequent to cuproptosis, is currently under investigation for its mechanisms in oncological contexts. In-depth analyses exploring the relationship between disulfidptosis-related genes (DRGs) and hepatocellular carcinoma (HCC) are currently limited.MethodsTranscriptomic data and clinical information were retrieved from the TCGA and GEO databases (GSE76427 and GSE54236), concentrating on the expression levels of 24 DRGs. Subsequently, multifactor and LASSO regression analyses were utilized to construct the 5-DRG prognostic signature. Immunohistochemistry (IHC) was employed to assess Cyclin A2 (CCNA2) protein expression levels. Quantitative real-time PCR (qRT-PCR) and western blot analyses were conducted to detect transcriptomic and protein expression of CCNA2-targeting short interfering RNA (siRNA). The Cell Counting Kit-8 (CCK-8) assay, EdU staining, and scratch experiments were employed to observe the proliferation and migration of hepatoma cell lines subsequent to CCNA2 inhibition.ResultsThree HCC patterns were identified, among which pattern B exhibited the the most unfavorable survival outcomes. Five DRGs (STC2, PBK, CCNA2, SERPINE1, and SLC6A1) were involved to establish the 5-DRG prognostic signature. High-risk groups (HRGs) exhibited prolonged survival durations in comparison to low-risk groups (LRGs). Both bioinformatics analyses and experimental methodologies corroborated the association of CCNA2 with poor prognosis in HCC patients. Functional studies elucidated that interference with CCNA2 significantly inhibited proliferation and migration, while simultaneously promoting apoptosis in hepatoma cells and resulting in the downregulation of epithelial-mesenchymal transition (EMT)-related protein markers.ConclusionsThe 5-DRG prognostic signature is proficient in predicting clinical outcomes, informing therapeutic strategies, and elucidating the characteristics of the immune microenvironment in HCC patients. Furthermore, this study elucidates the potential of CCNA2 as an innovative biomarker for HCC.

CCL4L2 participates in tendinopathy progression by promoting macrophage inflammatory responses: a single-cell analysis

BackgroundTendinopathy is very common in clinical practice, which is highly prevalent in athletes, sports enthusiasts and other people involved in high-load weight-bearing activities. Common types of tendinopathy include rotator cuff injury, Achilles tendinitis, tennis elbow and so on. Macrophages (Macs) are key immune cells in the pathogenesis of tendinopathy. In this study, CCL4L2+ M1-related signaling pathways were screened by combining single-cell RNA sequencing (scRNA-seq) to explore their significance in tendinopathy treatment.MethodsImmune cell populations were screened by Uniform Manifold Approximation and Projection (UMAP) downscaling, and Mac cell subsets were annotated using cell marker genes. The cellular communication mechanism between different cellular subsets such as Macs and tendon stem/progenitor cells (TSPCs) was demonstrated by cellular communication analysis. Based on cell marker genes of CCL4L2 + M1, Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment were performed to compare the expression differences in M1 and M2 between the Disease and Healthy groups. Associations between CCL4L2+ M1 and TSPCs were inferred by cell-cell communication analysis. The effects of CCL4L2 on Mac polarization and TSPCs were verified by enzyme-linked immunosorbent assay (ELISA) and real-time fluorescence quantitative PCR (qPCR).ResultsThe proportions of TSPCs, endothelial cells (ECs), smooth muscle cells (SMCs), and immune cells were significantly elevated in the Disease group. The proportion of M1 cells in the Disease group was higher than that in the Healthy group, while the proportion of M2 cells was lower than that in the Healthy group. M1 differentially expressed genes (DEGs) were mainly enriched to disease-related and immunoinflammation-related signaling pathways. Signaling intensities between M1 and TSPCs in pathways related to immunoinflammation and ischemic injury were significantly increased in the Disease group. The proportion of CCL4L2 + M1 in the Disease group was significantly higher than in the Healthy group, and communications between CCL4L2 + M1 and TSPCs varied significantly. Compared with the Control group, the expression levels of inflammatory cytokines were higher in the CCL4L2 group, and the expression levels of tendon differentiation markers (Egr1, Mkx, Scx, Type 1 collagen, Tnmd) were significantly down-regulated.ConclusionThe present study analyzed the heterogeneous alterations in the Healthy and Disease groups by scRNA-seq data and found that there was a significant inflammatory infiltrate in the Disease group with markedly increased Mac activity, which was associated with activation of the CCL4L2 + M1-associated signaling pathways. CCL4L2 promotes M1 polarization and inhibits TSPC differentiation through activating M1-related inflammatory signaling pathways. These findings contribute to a more comprehensive understanding of tendon injury progression and provide potential targets for tendinopathy treatment.

Development and application of a quadruple RT-qPCR assay for the simultaneous detection of NoV GI, NoV GII, and HAV in bivalve shellfish.

To achieve rapid and simultaneous detection of NoV GI, NoV GII, and HAV, a quadruple real-time fluorescence quantitative PCR (RT-qPCR) assay was developed using MS2 bacteriophage as a process control virus. The quadruple RT-qPCR assay effectively detected NoV GI, NoV GII, HAV, and MS2 RNA with detection limits of 102 copies/μL, 103 copies/μL, 102 copies/μL, and 103 copies/μL, respectively, within 1 hour 50 minutes. The quadruple RT-qPCR assay could specifically detect NoV GI, NoV GII, HAV, and MS2 without cross-reactions with other common pathogens, demonstrating good reproducibility with intra-assay and inter-assay coefficients of variation all below 2.11%. In this study, 337 bivalve shellfish samples collected from various regions of Hebei Province were pretreated using the proteinase K-PEG 8000 precipitation-chloroform method, and viral nucleic acids were enriched and extracted from a volume of viral solution that was doubled. The developed quadruple RT-qPCR assay was used to detect NoV GI, NoV GII, and HAV in bivalve shellfish samples, and the positive rates were 19.88% (67/337), 20.47% (69/337), and 4.75% (16/337), respectively. In addition, mixed infections of NoV GI and NoV GII (10.68%, 36/337) and NoV GI and HAV (0.89%, 3/337) were observed. In all, 200 bivalve shellfish samples were randomly selected for the assay, and it was found that the total, positive, negative coincidence rates, and Kappa values of the quadruple RT-qPCR assay were 98.3%, 99.1%, 98.2%, and 0.945, respectively, compared with the single RT-qPCR assay. These results show that the developed quadruple RT-qPCR assay has comparable performance to the single RT-qPCR assay.IMPORTANCEFood-borne diseases caused by viral contamination have become a growing concern, and bivalve shellfish is a crucial source of infection, with many outbreaks of non-bacterial acute gastroenteritis associated with raw food or the use of undercooked shellfish such as oysters. As food contamination problems caused by NoV and HAV become more severe, it is important to study and establish a sensitive and efficient assay to simultaneously detect NoV and HAV by applying the MS2 process control virus for the protection of bivalve shellfish food safety and the monitoring of the above food-borne viral contamination. In addition, bivalve shellfish samples contain a large number of PCR inhibitors such as polysaccharides, lipids, and proteins, so optimization of the virus enrichment and extraction method is essential and is expected to provide a research basis for subsequent related experiments.

Integrating bioinformatics and machine learning to unravel shared mechanisms and biomarkers in chronic obstructive pulmonary disease and type 2 diabetes.

Chronic obstructive pulmonary disease (COPD) and type 2 diabetes mellitus (T2DM) are on the rise. While there is evidence of a link between the two diseases, the pathophysiological mechanisms they share are not fully understood. In this study, the co-expressed genes of COPD and T2DM in Gene Expression Omnibus database were identified by bioinformatics method, and the functional enrichment analysis was performed. Machine learning algorithms were used to identify biomarkers. The diagnostic value of these biomarkers was assessed by receiver operating characteristic analysis, and their relationship to immune cells was investigated by immunoinfiltration analysis. Finally, real-time quantitative polymerase chain reaction was performed. A total of five overlapping genes were obtained, focusing on pathways associated with insulin resistance and inflammatory mediators. The machine learning method identified three biomarkers: matrix metalloproteinase 9, laminin α4, and differentially expressed in normal cells and neoplasia domain containing 4 C, all of which were shown to have high diagnostic values by receiver operating characteristic analysis. Immunoinfiltration analysis showed that it was associated with a variety of immune cells. In addition, the real-time quantitative polymerase chain reaction results confirmed agreement with our bioinformatics analysis. Our study sheds light on the common pathogenesis and biomarkers of both diseases, and these findings have potential implications for the development of new diagnostic and treatment strategies for COPD and T2DM. Key message What is already known on this topic? Chronic obstructive pulmonary disease (COPD) and type 2 diabetes mellitus (T2DM) often coexist as comorbidities. However, the exact mechanistic link between the two diseases remains complex, multifactorial, and not fully understood. What this study adds? Three biomarkers, including matrix metalloproteinase, laminin α4, and differentially expressed in normal cells and neoplasia domain containing 4 C, were identified as key co-expression hub genes in COPD and T2DM. How this study might affect research, practice or policy? Future studies may benefit from incorporating a larger sample set to further explore and validate the diagnostic and therapeutic effects of these core genes.

Developing the new diagnostic model by integrating bioinformatics and machine learning for osteoarthritis

BackgroundOsteoarthritis (OA) is a common cause of disability among the elderly, profoundly affecting quality of life. This study aims to leverage bioinformatics and machine learning to develop an artificial neural network (ANN) model for diagnosing OA, providing new avenues for early diagnosis and treatment.MethodsFrom the Gene Expression Omnibus (GEO) database, we first obtained OA synovial tissue microarray datasets. Differentially expressed genes (DEGs) associated with OA were identified through utilization of the Limma package and weighted gene co-expression network analysis (WGCNA). Subsequently, protein-protein interaction (PPI) network analysis and machine learning were employed to identify the most relevant potential feature genes of OA, and ANN diagnostic model and receiver operating characteristic (ROC) curve were constructed to evaluate the diagnostic performance of the model. In addition, the expression levels of the feature genes were verified using real-time quantitative polymerase chain reaction (qRT-PCR). Finally, immune cell infiltration analysis was performed using CIBERSORT algorithm to explore the correlation between feature genes and immune cells.ResultsThe Limma package and WGCNA identified a total of 72 DEGs related to OA, of which 12 were up-regulated and 60 were down-regulated. Then, the PPI network analysis identified 21 hub genes, and three machine learning algorithms finally screened four feature genes (BTG2, CALML4, DUSP5, and GADD45B). The ANN diagnostic model was constructed based on these four feature genes. The AUC of the training set was 0.942, and the AUC of the validation set was 0.850. In addition, the qRT-PCR validation results demonstrated a significant downregulation of BTG2, DUSP5, and GADD45 mRNA expression levels in OA samples compared to normal samples, while CALML4 mRNA expression level exhibited an upregulation. Immune cell infiltration analysis revealed B cells memory, T cells gamma delta, B cells naive, Plasma cells, T cells CD4 memory resting, and NK cells The abnormal infiltration of activated cells may be related to the progression of OA.ConclusionsBTG2, CALML4, DUSP5, and GADD45B were identified as potential feature genes for OA, and an ANN diagnostic model with good diagnostic performance was developed, providing a new perspective for the early diagnosis and personalized treatment of OA.

TFAP2A Promotes Cell Progression and Suppresses Ferroptosis in Lung Adenocarcinoma via Activating Transcription of CST1.

Lung adenocarcinoma (LUAD) is a common type of lung cancer with complicated pathological mechanism. Transcription Factor AP-2 Alpha (TFAP2A) and Cysteine protease inhibitor 1 (CST1) are upregulated genes in LUAD samples, accordingly, we focused on clarifying the role of TFAP2A/CST1 axis in LUAD. Expression analysis was performed using real-time quantitative polymerase chain reaction and western blot. Cellular behaviors were detected by colony formation assay, EdU assay, wound healing assay and flow cytometry. Ferroptosis was assessed by oxidative indicators, Fe2+ level and related proteins. TFAP2A and CST1 interaction was analyzed via ChIP assay and dual-luciferase reporter assay. TFAP2A function in vivo was evaluated by xenograft tumor assay. CST1 was overexpressed in LUAD samples and cells. Downregulation of CST1 inhibited proliferation, migration but it promoted apoptosis and ferroptosis of LUAD cells. TFAP2A interacted with the promoter of CST1 to up-regulate CST1 expression. TFAP2A regulated the malignant behaviors and ferroptosis of LUAD cells by targeting CST1. TFAP2A affected LUAD tumor growth via mediating CST1. All these data proved that TFAP2A/CST1 axis contributed to proliferation, migration while it suppressed apoptosis and ferroptosis in LUAD.

Detection of transmembrane protein 100 in breast cancer: Correlation with malignant progression and chemosensitivity

Objective: With increased incidence, breast cancer has become the most common malignant tumor in women. Transmembrane protein 100 (TMEM100) is a key factor affecting the progression of malignant tumors. The aim of the study is to examine the molecular mechanism of TMEM100 in malignant progression. Material and Methods: TMEM100 expression was analyzed by Western blot, immunohistochemistry, and real-time quantitative polymerase chain reaction. Cell migration and invasiveness after transfection with TMEM100 were investigated by Transwell assay. 5-ethynyl-2-deoxyuridine staining and cell colony-formation assay were utilized to the exploration of cell proliferation. Flow cytometry was adopted to detect whether TMEM100 affected the effect of Docetaxel on cell apoptosis. The effects of TMEM100 on the Ras-extracellular signal-regulated kinase (RAS/ERK) pathway were explored by Western blot assay. Results: Downregulated TMEM100 expression was in breast cancer tissues (P < 0.01). TMEM100 overexpression hindered the invasion (P < 0.01), migration (P < 0.01), and proliferation (P < 0.01) of breast cancer cells. Chemotherapy sensitivity of breast cancer cells to docetaxel was enhanced by TMEM100 (P < 0.01). TMEM100 inhibited Ras expression and ERK1/2 phosphorylation (P < 0.01). Furthermore, ERK agonist TertButylhydroquinone neutralized the effects of TMEM100 (P < 0.01). Conclusion: TMEM100 blocked malignant progression of breast cancer and enhanced docetaxel chemosensitivity by suppressing RAS/ERK pathway. These data manifested that regulation of TMEM100 expression may affect the progression of breast cancer, and its prognostic value and mechanism deserve further investigation.

Energy restriction inhibits β-catenin ubiquitination to improve ischemic stroke injury via USP18/SKP2 axis.

Ischemic stroke (IS) remains a global health issue because of its great disability and mortality. Energy restriction (ER) has been justified to perform an inhibitory role in cerebral injury caused by IS. This research was purposed to inquire the potential molecular mechanism of ER in IS. To verify the function of ER in the animal and cell models of IS, rats were subjected to intermittent fasting (IF) and middle cerebral artery occlusion/reperfusion (MCAO/R) surgery and HAPI cells were treated with oxygen-glucose deprivation and reoxygenation (OGD/R) and 2-deoxyglucose (2-DG). It was disclosed that IF mitigated brain damage and inflammation in MCAO/R rats. Likewise, ER inhibited OGD/R-evoked microglial activation and inflammatory response. Of note, ubiquitin specific protease 18 (USP18) was uncovered to be the most significantly upregulated in MCAO/R rats receiving IF compared to free-feeding MCAO/R rats. Real-time quantitative polymerase chain reaction (RT-qPCR) and western blot verified that ER led to the promotion of USP18 expression. Moreover, downregulation of USP18 neutralized the meliorative effects of ER on OGD/R-treated HAPI cells. Functionally, USP18 restrained β-catenin ubiquitination to enhance its expression. In addition, our results manifested that S-phase kinase associated protein 2 (SKP2) contributed to degradation of β-catenin and USP18 abolished the role of SKP2 in β-catenin ubiquitination. Knockout of USP18 eliminated the protective effects of IF on MCAO/R rats, while SKP2 exacerbated brain damage and inflammation by decreasing β-catenin expression after IF. In summary, we validated that ER-induced USP18 exerts a suppressive function in IS damage through SKP2-mediated β-catenin ubiquitination.

MiR-484 as an "OncomiR" in Breast Cancer Promotes Tumorigenesis by Suppressing Apoptosis Genes.

Breast cancer (BC) is one of the most common causes of death among females. Cancer cells escape from apoptosis, causing the cells to proliferate uncontrollably. MicroRNAs (miRNAs) are known to regulate apoptosis in cancer cells. This study aimed to determine the change in miR-484 in different BC cells and its relationship with the apoptosis pathway. In the study, tumor and healthy tissue samples adjacent to the tumor were collected from 42 patients (6 benign, 36 malignant). Tissue samples were classified according to tumor type, tumor histological grade, proliferation index, and molecular subtypes. Gene expression levels were determined by quantitative real-time polymerase chain reaction (qRT-PCR), and protein levels were determined using the Western Blot method. The results were analyzed using the delta-delta Ct method. Findings showed that miR-484 expression levels were higher in malignant tumors than in benign tumors, and higher in tumor tissues than healthy tissues. Additionally, it was determined that as Ki-67 levels and histological grade and aggressiveness increased, miR-484 expression levels also increased. In tumor tissue compared with healthy adjacent tissue, there was an increase in BCL2 expression and a decrease in Casp3 and Casp9 expression. Therefore, a positive correlation was found between miR-484 expression and BCL2, and a negative correlation was found between CASP3 and CASP9 expression. Our results show that miR-484 may play a roll as an onco-miR in BC. Increased miR-484 and BCL2, and decreased Casp3, in breast tumor tissues suggest that Casp9 expression may increase uncontrolled cell proliferation by suppressing apoptosis in BC cells and may contribute to tumor progression.

MiR-3191 promotes the proliferation and metastasis of hepatocellular carcinoma via regulating PAK6

Background/AimsmicroRNAs (miRNAs) contribute to tumorigenesis, progression and drug resistance of hepatocellular carcinoma (HCC). miR-3191 is a newly discovered miRNA, and its function and mechanism of action in biological processes and diseases are not completely understood.MethodsmiR-3191 expression is determined via quantitative real-time polymerase chain reaction. Knockdown and overexpression of miR-3191 influence the proliferation and metastasis of HCC cells, which is measured by Cell Counting Kit-8 assay, Colony Formation assay and Cell metastasis assay. Protein expression is estimated by Western blot. The interplay between miR-3191 and target is validated by dual-luciferase reporter assay.ResultsHere, we show that miR-3191 is upregulated in HCC tissues and associated with poor prognosis of HCC patients. Mechanistically, p21-activated protein kinase 6 (PAK6) was identified as a direct target of miR‑3191 in HCC. PAK6 knockdown partially recovered interference of miR‑3191‑induced decrease in cell proliferation and invasion. The accuracy of HCC patient prognosis could be improved by employing a combination of miR-3191 and PAK6 values.ConclusionsmiR-3191 promotes the proliferation and metastasis of HCC cells via targeting PAK6 and may serve as a prognostic biomarker and potential therapeutic target.

Analysis of Fibroblast Growth Factor 2 Impact and Mechanism on Broncho-Pulmonary Dysplasia.

Epithelial-mesenchymal transition (EMT) of alveolar epithelial cells is an important mechanism for the onset and development of broncho-pulmonary dysplasia (BPD).The role of FGF-2 in BPD is currently unclear. The aim of our study is to investigate the expression of FGF-2 in lung tissue of BPD mice, to further clarify the effect of FGF-2 on EMT in alveolar epithelial cells and to actively search for possible signaling pathways. The BPD model was induced by exposure to hyperoxia. Lung tissue samples were collected and Hematoxylin and eosin (HE) staining was used to determine the modelling effect. Quantitative Real-time Polymerase Chain Reaction (QRT-PCR), immunohistochemistry were used to detect FGF-2 expression in BPD mice. To further investigate the effect of FGF-2 supplementation and deficiency on EMT in alveolar epithelial cells, A549 cells were cryopreserved, resuspended, cultured and passaged. Transforming growth factor-β1 (TGFβ1) was used to induce EMT. FGF-2 small interfering RNA fragments were synthesised and screened. Fbroblast growth factor receptor1 (FGFR1) expression was inhibited by BGJ398. (3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H tetrazolium) (MTS) assay was used to detect the effect of FGF-2 and Infigratinib (BGJ398) on cell proliferation. We used qRT-PCR and Western blot to detect the expression of epithelial cell markers, mesenchymal cell markers and EMT-related signaling pathway proteins. Our results showed that the successful established hyperoxia mice model were characteristic by BPD. Hyperoxia decreased FGF-2 on day 4, upregulated FGF-2 on day 21, which resulted in EMT. In vitro, we found that FGF-2 alone increased the expression of mesenchymal markers, decreased the expression of epithelial markers and activated Phosphatidylinositol 3-kinase/Protein kinase B (PI3K/AKT), Small mother against decapentaplegic (Smad), mitogen-activated protein kinase (P38) and extracellular signal-regulated kinase (ERK) signaling pathways. FGF-2 could not reverse but synergistically promote TGF-β1-induced EMT of alveolar epithelial cells. Silencing FGF-2 increased the expression of epithelial marker E-cadherin, inhibited the PI3K/AKT, Smad, and P38 signaling pathways activated by TGF-β1, but activated ERK signaling. FGF-2 receptor inhibitor BGJ398 reversed TGF-β1-induced EMT, decreased the expression of FGFR1, and inhibited ERK signaling pathway activation. FGF2 was closely associated with EMT in BPD mice. Both high and low levels of FGF2 promoted EMT in A549. The FGF-2 receptor inhibitor BGJ398 reversed TGF-β1-induced EMT in A549 by inhibiting the FGFR1/P-ERK signaling pathway.

Prognostic feature based on androgen-responsive genes in bladder cancer and screening for potential targeted drugs

ObjectivesBladder cancer (BLCA) is a tumor that affects men more than women. The biological function and prognostic value of androgen-responsive genes (ARGs) in BLCA are currently unknown. To address this, we established an androgen signature to determine the prognosis of BLCA.MethodsSequencing data for BLCA from the TCGA and GEO datasets were used for research. The tumor microenvironment (TME) was measured using Cibersort and ssGSEA. Prognosis-related genes were identified and a risk score model was constructed using univariate Cox regression, LASSO regression, and multivariate Cox regression. Drug sensitivity analysis was performed using Genomics of drug sensitivity in cancer (GDSC). Real-time quantitative PCR was performed to assess the expression of representative genes in clinical samples.ResultsARGs (especially the CDK6, FADS1, PGM3, SCD, PTK2B, and TPD52) might regulate the progression of BLCA. The different expression patterns of ARGs may lead to different immune cell infiltration. The risk model indicates that patients with higher risk scores have a poorer prognosis, more stromal infiltration, and an enrichment of biological functions. Single-cell RNA analysis, bulk RNA data, and PCR analysis support the reliability of this risk model, and a nomogram was also established for clinical use. Drug prediction analysis showed that high-risk patients had a better response to fludarabine, AZD8186, and carmustine.ConclusionARGs played an important role in the progression, immune infiltration, and prognosis of BLCA. The ARGs model has high accuracy in predicting the prognosis of BLCA patients and provides more effective medication guidelines.

Overcoming the Silencing of Doxycycline-Inducible Promoters in hiPSC-derived Cardiomyocytes

Background Human induced pluripotent stem cells (hiPSCs) are pivotal for studying human development, modeling diseases, and advancing regenerative medicine. Effective control of transgene expression is crucial to achieve temporal and quantitative precision in all of these contexts. The doxycycline (dox)-inducible OPTi-OX system, which integrates the Tet-On 3G transactivator and dox-responsive transgene at the hROSA26 and AAVS1 genomic safe harbors (GSHs), respectively, offers a promising solution. Yet, transgene silencing, particularly in hiPSC-derived cardiomyocytes (hiPSC-CMs), limits its utility. Methods To address this, we evaluated strategies to enhance dox-inducible transgene expression. We compared two promoters, TRE3VG and T11, for activity and stability, and investigated the addition of a Ubiquitous Chromatin Opening Element (UCOE) to reduce silencing. We also tested relocating the transgene cassette to the CLYBL GSH, and employed sodium butyrate (SB), a histone deacetylase inhibitor, to restore promoter activity. Transgene expression was assessed via flow cytometry and real-time quantitative PCR. Results TRE3VG exhibited higher activity than T11, but both were prone to silencing. UCOE did not enhance promoter activity in hiPSCs, but modestly reduced silencing in hiPSC-CMs. Targeting the CLYBL locus improved promoter activity compared to AAVS1 in both hiPSCs and hiPSC-CMs. SB restored activity in silenced inducible promoters within hiPSC-CMs, but compromised hiPSC viability. Unexpectedly, Tet-On 3G was silenced in some clones and could not be reactivated by SB. Conclusions These findings underscore the need for integrating multiple strategies, including careful GSH selection, improved cassette design, epigenetic modulation, and clone screening, to develop robust dox-inducible systems that retain functionality during hiPSC differentiation.

Chitosan-casein as novel drug delivery system for transferring Phyllanthus emblica to inhibit Pseudomonas aeruginosa

This study investigated the ability of Phyllanthus emblica encapsulated within chitosan-coated casein (CS-casein-Amla) nanoparticles to inhibit the growth of multi-drug-resistant Pseudomonas aeruginosa (P. aeruginosa) bacteria and prevent the formation of biofilms. The MDR strains underwent screening, and the morphological characteristics of the resulting nanoparticles were assessed using SEM, DLS, and FTIR. In addition, the efficacy of encapsulation, stability, and drug release were evaluated. The PpgL, BdlA, and GacA biofilm gene transcription quantities were quantified by quantitative real-time PCR. Simultaneously, the nanoparticles were assessed for their antibacterial and cytotoxic effects using the well diffusion and MTT procedures. CS-casein-Amla nanoparticles with a size of 500.73 ± 13 nm, encapsulation efficiency of 76.33 ± 0.81%, and stability for 60 days at 4 °C (Humidity 30%) were created. The biological analysis revealed that CS-casein-Amla nanoparticles exhibited strong antibacterial properties. This was shown by their capacity to markedly reduce the transcription of PpgL, BdlA, and GacA biofilm genes at a statistically significant value of p ≤ 0.01. The nanoparticles demonstrated decreased antibiotic resistance compared to unbound Amla and CS-casein. Compared to Amla, CS-casein-Amla nanoparticles showed very little toxicity against HDF cells at dosages ranging from 1.56 to 100 µg/mL (p ≤ 0.01). The results highlight the potential of CS-casein-Amla nanoparticles as a significant advancement in combating highly resistant P. aeruginosa. The powerful antibacterial properties of CS-casein-Amla nanoparticles against P. aeruginosa MDR strains, which are highly resistant pathogens of great concern, may catalyze the development of novel antibacterial research approaches.

Promoter of Vegetable Pea PsPIP2-4 Responds to Abiotic Stresses in Transgenic Tobacco

Plasma membrane intrinsic proteins (PIPs), one sub-family of aquaporins (AQPs), are responsible for plant abiotic stress responses. However, little information is currently available about the stress responsiveness of the PIP promoter in vegetable pea. In the present study, one novel promoter of PsPIP2-4 which shared high similarity to the PIP2-type AQPs from other plants, was isolated. Quantitative real-time PCR (qRT-PCR) assays suggested that PsPIP2-4 was predominantly expressed in leaves and abundantly induced by abiotic stress treatments (polyethylene glycol (PEG) 6000, NaCl, and methyl jasmonate (MeJA)). Further, the promoter activity of PsPIP2-4 was verified in transgenic tobacco plants. Beta-glucuronidase (GUS) staining driven by the PsPIP2-4 promoter confirmed that it was mainly detected in the leaves of transgenic seedlings, especially in the guard cells. Exposure of transgenic seedlings to various environmental stimuli proved that the promoter activity of PsPIP2-4 was abundantly strengthened by osmotic, salt, and MeJA stresses. This research provides one stress-inducible promoter enabling targeted gene expression under abiotic stresses and demonstrates its usefulness in the genetic improvement of plant stress resistance.

LEP O-GlcNAcylation inactivates NF-κB pathway by suppressing LEP protein level and thus mediates cellular senescence and osteogenic differentiation in mouse mesenchymal stem cells

BackgroundCellular senescence is a key driver of decreased bone formation and osteoporosis. Leptin (LEP) has been implicated in cellular senescence and osteogenic differentiation. The aim of this study was to investigate the mechanisms by which LEP mediates cellular senescence and osteogenic differentiation.MethodsC3H10T1/2 cells were treated with etoposide to induce cellular senescence, which was assessed by β-galactosidase staining. Quantitative real-time PCR and western blotting were used to measure the levels of senescence markers p21 and p16, as well as osteogenic differentiation-related genes ALP, COL1A1, and RUNX2. Alkaline phosphatase (ALP) staining and alizarin red S staining were performed to evaluate osteogenic differentiation. The NF-κB pathway and O-GlcNAcylation were assessed by western blotting.ResultsEtoposide treatment increased the number of senescent cells and the levels of p21 and p16, along with elevated LEP expression. These effects were reversed by LEP knockdown. Additionally, LEP knockdown increased ALP staining density and osteoblast mineralization nodules, as well as the mRNA and protein levels of ALP, COL1A1, and RUNX2, indicating that LEP knockdown promoted osteogenic differentiation in C3H10T1/2 cells. Mechanistically, LEP knockdown inactivated the NF-κB pathway by inhibiting the nuclear translocation of p65. Furthermore, OGT was found to promote O-GlcNAcylation of LEP at the S50 site.ConclusionOur findings demonstrated that O-GlcNAcylation of LEP inactivated the NF-κB pathway by reducing LEP protein levels, thereby inhibiting cellular senescence and promoting osteogenic differentiation in C3H10T1/2 cells. This study may provide a novel therapeutic target for the treatment of osteoporosis.

Cannabichromene as a Novel Inhibitor of Th2 Cytokine and JAK/STAT Pathway Activation in Atopic Dermatitis Models

Cannabichromene (CBC) is one of the main cannabinoids found in the cannabis plant, and although less well known than tetrahydrocannabinol (THC) and cannabidiol (CBD), it is gaining attention for its potential therapeutic benefits. To date, CBC’s known mechanisms of action include anti-inflammatory, analgesic, antidepressant, antimicrobial, neuroprotective, and anti-acne effects through TRP channel activation and the inhibition of inflammatory pathways, suggesting that it may have therapeutic potential in the treatment of inflammatory skin diseases, such as atopic dermatitis (AD), but its exact mechanism of action remains unclear. Therefore, in this study, we investigated the effects of CBC on Th2 cytokines along with the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathways involved in AD pathogenesis. We used a 2,4-Dinitrochlorobenzene (DNCB)-induced BALB/c mouse model to topically administer CBC (0.1 mg/kg or 1 mg/kg). The results showed that skin lesion severity, ear thickness, epithelial thickness of dorsal and ear skin, and mast cell infiltration were significantly reduced in the 0.1 mg/kg CBC-treated group compared with the DNCB-treated group (p < 0.001). In addition, real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis showed a significant decrease in the mRNA expression of Th2 cytokines (TSLP, IL-4, IL-13) and inflammatory mediators (IFN-γ, IL-1β, IL-6, IL-17, IL-18, and IL-33) (p < 0.05). Western blot analysis also revealed a significant decrease in JAK1, JAK2, STAT1, STAT2, STAT3, and STAT6 protein expression (p < 0.05). These results suggest that CBC is a promising candidate for the treatment of AD and demonstrates the potential to alleviate AD symptoms by suppressing the Th2 immune response.