Quantitative Reverse Transcription-polymerase Chain Reaction Research Articles

Photobiomodulation Promotes Early Recovery of Olfactory Function and Modulates Neuroprotective Gene Expression in a Mouse Model of Ischemic Stroke.

Ischemic stroke often leads to neurological deficits, including olfactory dysfunction, which can significantly diminish quality of life. Photobiomodulation (PBM) has emerged as a promising therapeutic strategy for enhancing post-stroke recovery, although the molecular mechanisms, particularly regarding gene expression change, are not yet fully understood. This study investigates the long-term effects of photothrombosis (PT) on olfactory function and the olfactory bulb (OB) microenvironment, with a focus on PBM's efficacy during both early and late phases. In a mouse OB PT stroke model, PBM therapy (808-nm laser, 40J/cm2 fluence, 325 mW/cm2, 2min daily) was applied from day 2 to day 7 post-PT. Olfactory function was monitored from pre-stroke through day 28 using the buried food test (BFT), and MRI scans were performed on days 7 and 28 to assess tissue damage. RNA sequencing (RNA-seq) and reverse transcription quantitative PCR (RT-qPCR) were conducted on day 7 to evaluate gene expression changes, with additional RT-qPCR analyses performed on day 28. PBM significantly accelerated olfactory function recovery by day 14, with full recovery maintained through day 28. Despite functional recovery, MRI results indicated persistent infarction at 28days. RNA-seq identified upregulation of neuroprotective genes, including Gpr39 and Or4m1, following PBM treatment, suggesting enhanced gene expression related to acute-phase recovery. However, the impact of PBM on gene expression and functional recovery appeared to wane in the later stages of recovery. These findings underscore PBM's potential to enhance early-stage recovery in ischemic stroke, though its benefits may be more limited in the chronic phase.

Assessment of the transmission of live-attenuated chikungunya virus vaccine VLA1553 by Aedes albopictus mosquitoes

BackgroundChikungunya virus (CHIKV) is a mosquito-transmitted, arthritogenic alphavirus that causes sporadic outbreaks of often debilitating rheumatic disease. The recently approved CHIKV vaccine, IXCHIQ, is based on a live-attenuated CHIKV strain (VLA1553), with viraemic vaccine recipients theoretically able to transmit VLA1553 to mosquitoes with ensuing onward transmission. We thus evaluated VLA1553 transmission from artificial blood meals to Aedes albopictus mosquitoes, and onward transmission to mice.MethodsFemale A. albopictus mosquitoes were fed on defibrinated sheep blood containing wild-type CHIKV (viral titre: 7.50 log10CCID50/mL) or VLA1553 (viral titres: 7.85, 5.72, 4.58, and 3.79 log10CCID50/mL). Viral titres in mosquito bodies and saliva were determined using CCID50 assays 7–8 days after the blood meal. After providing CHIKV or VLA1553 (viral titres ~ 7–8 log10CCID50/mL) in blood meals to mosquitoes, infected mosquitoes were fed on highly susceptible Irf3/7−/− mice (n = 3 per group). Data were re-analysed using the same reverse transcription quantitative polymerase chain reaction (RT-qPCR) as for an earlier VLA1553 phase 1 clinical trial, to allow correlations between blood meal titres and viraemia in vaccine recipients.ResultsMosquito body viral titres were significantly higher (P < 0.0001) for CHIKV versus VLA1553-fed mosquitoes at blood meal viral titres of ~ 7–8 log10CCID50/mL. Mosquito body VLA1553 titres decreased with reducing blood meal titres, but there was no dose-dependent effect on saliva viral titres. No dissemination to salivary glands was seen at blood meal titres ≤ 3.875 log10CCID50/mL. CHIKV-fed mosquitoes were able to transmit virus, and induce viraemia in, 3/3 Irf3/7−/− mice via mosquito bites. In contrast, 0/3 Irf3/7−/− mice became infected after bites from VLA1553-fed mosquitoes. RT-qPCR comparisons with phase 1 clinical data for VLA1553-vaccinated individuals indicated that VLA1553 viraemia was at or below the aforementioned threshold for transmission.ConclusionsThe evidence presented herein argue that the low viraemia in VLA1553-vaccinated individuals would mitigate against transmission. In addition, replication of VLA1553 in mosquito bodies was also significantly attenuated. Overall, mosquito-borne transmission of VLA1553 from vaccinated individuals to others appears improbable.Graphical

Transcriptomics-based exploration of ubiquitination-related biomarkers and potential molecular mechanisms in laryngeal squamous cell carcinoma

BackgroundOne of the most common and prevalent cancers is laryngeal squamous cell carcinoma (LSCC), which poses a great threat to the life and health of the patient. Nonetheless, it has been demonstrated that ubiquitination is crucial for the development and course of LSCC. Therefore, it is particularly important to identify biomarkers for ubiquitination-related genes (UbRGs) in LSCC.MethodsDifferentially expressed genes (DEGs) in the LSCC versus controls were obtained by differential expression analysis. Also, key modular genes associated with LSCC were obtained using weighted gene co-expression network analysis (WGCNA). Next, DEGs, key module genes, and UbRGs were taken to intersect to obtain candidate genes. And then machine algorithms were to screen potential biomarkers, further their diagnostic value were analyzed and validated. Then, therapeutic agents for biomarkers were predict. In addition, the regulatory networks of the biomarkers were mapped. The expression levels of biomarkers were detected in clinical samples using reverse transcription-quantitative PCR (RT-qPCR).ResultsA total of eight candidate genes were acquired by the overlap 1,911 DEGs, the key modular genes of WGCNA, and 1,393 UbRGs. A sum of four biomarkers (WDR54, KAT2B, NBEAL2 and LNX1) were identified by two machine learning, then these four biomarkers were validated in GSE127165 and the expression trend was consistent with TCGA-LSCC, they were recorded as biomarkers. Moreover, the accuracy of the biomarkers in predicting clinical aspects of LSCC was confirmed by the receiver operating characteristic (ROC) curves. Subsequently, cancers such as malignant neoplasms, colorectal cancers, tumors, and primary malignant neoplasms were significantly associated with the biomarkers, which further suggests that these four biomarkers were strongly associated with cancer. Meanwhile, the drugs garcinol, cocaine, and triazolam, among others, used for LSCC treatment were predicted. Finally, transcription factors (TFs) (BRD4, MYC, AR, and CTCF) were predicted to regulate the biomarkers. RT-qPCR assays illustrated that the expression trends of KAT2B, LNX1 and NBEAL2 remained consistent with the dataset.ConclusionThe identification of four biomarkers (WDR54, KAT2B, NBEAL2 and LNX1) associated with UbRGs could ultimately serve as a predictive clinical diagnosis of LSCC and provide insight into the molecular mechanisms of LSCC.

Mechanistic insights into PDZK1-interacting protein 1 on the malignant progression of colorectal carcinoma

Objective: PDZ domain containing 1-interacting protein 1 (PDZK1IP1) is commonly overexpressed in a wide variety of cancer. Hence, the objective of the present study is to ascertain the influences of PDZK1IP1 on colorectal carcinoma (CRC) development. Material and Methods: PDZK1IP1 expression was tested through reverse transcription-quantitative polymerase chain reaction and Western blot analysis, and its correlation with prognosis was analyzed using the GEPIA website. Small interfering RNA against PDZK1IP1 was adopted to downregulate PDZK1IP1 expression in CRC cells. The effects of PDZK1IP1 on cell growth were ascertained using colony formation and CCK-8 tests, and CRC cell apoptosis was analyzed through flow cytometry. Cell migration capability and invasiveness were measured using Matrigel Transwell and scratch-healing assays. Results: PDZK1IP1 was highly expressed in the CRC tissues (P &lt; 0.001) and cells (P &lt; 0.05), and its knockdown restrained cell growth (P &lt; 0.05), migratory potential (P &lt; 0.01), and invasive capacities (P &lt; 0.001) and accelerated cell apoptosis (P &lt; 0.001). Mechanically, PDZK1IP1 silencing blocked CRC progression by inactivating the phosphatidylinositol 3-kinase/protein kinase B/mechanistic target of the rapamycin pathway. Conclusion: PDZK1IP1 contributes to the oncogenesis of CRC. This finding provides a basis for the diagnosis, treatment, and prevention of CRC.

UPP1 and AHSA1 as emerging biomarkers and targets in pancreatic cancer: A proteomic approach.

The specific protein targets involved in pancreatic cancer (PC) pathogenesis and its varying levels of differentiation remain incompletely understood. Advanced proteomic methodologies provide a powerful means of identifying key regulatory proteins and signaling pathways central to cancer progression. In this study, proteomic analyses were performed on PC tissue samples of different differentiation grades, along with adjacent non-cancerous (para-PC) tissues. Bioinformatics techniques were used to identify differentially expressed proteins (DEPs) and their associated pathways. Key target proteins were validated using the Gene Expression Profiling Interactive Analysis (GEPIA) database, reverse transcription-quantitative polymerase chain reaction (RT-qPCR), Western blotting, immunohistochemistry (IHC), and immunofluorescence (IF). A total of 431 DEPs were identified between PC and para-PC tissues, while 470 DEPs distinguished poorly differentiated (PD) from moderately differentiated (MD) PCs. Functional enrichment analysis revealed that these DEPs participate in various biological processes and signaling pathways. Five DEPs were common to both comparisons, with Uridine Phosphorylase 1 (UPP1), Lactamase Beta, and Activator of HSP90 ATPase Activity 1 (AHSA1) showing particularly notable differences. UPP1 and AHSA1 were significantly upregulated in PC tissues relative to adjacent tissues and exhibited even higher expression in PD-PCs compared to MD ones. These findings were consistently supported by GEPIA, RT-qPCR, Western blotting, IHC, and IF analyses. This study identifies UPP1 and AHSA1 as key proteins linked to PC differentiation and progression, highlighting their potential as diagnostic markers and therapeutic targets. These insights enhance our understanding of the molecular mechanisms underlying PC and open new avenues for precision treatment strategies.

Identification of genetic indicators linked to immunological infiltration in idiopathic pulmonary fibrosis.

This study employed bioinformatics to investigate potential molecular markers associated with idiopathic pulmonary fibrosis (IPF) and examined their correlation with immune-infiltrating cells. Microarray data for IPF were retrieved from the Gene Expression Omnibus database. Differentially expressed genes (DEGs) and module genes were identified through Limma analysis and weighted gene co-expression network analysis. Enrichment analysis and protein-protein interaction network development were performed on the DEGs. Machine learning algorithms, including least absolute shrinkage and selection operator regression, random forest, and extreme gradient boosting, were applied to identify potential key genes. The predictive accuracy was assessed through a nomogram and a receiver operating characteristic (ROC) curve. Additionally, the correlation between core genes and immune-infiltrating cells was assessed utilizing the CIBERSORT algorithm. An IPF model was established in a human fetal lung fibroblast 1 (HFL-1) through induction with transforming growth factor β1 (TGF-β1), and validation was conducted via reverse transcription-quantitative polymerase chain reaction. A sum of 1246 genes exhibited upregulation, whereas 879 genes were downregulated. Pathway enrichment analysis and functional annotation revealed that DEGs were predominantly involved in extracellular processes. Four key genes - cd19, cxcl13, fcrl5, and slamf7 - were identified. Furthermore, ROC analysis demonstrated high predictive accuracy for these 4 genes. Compared to healthy individuals, lung tissues from IPF patients exhibited an increased presence of plasma cells, CD4 memory-activated T cells, M0 macrophages, activated dendritic cells, resting NK cells, and M2 macrophage infiltration. The upregulation of cd19, cxcl13, fcrl5, and slamf7 in TGF-β1-treated HFL-1 cells was confirmed, aligning with the findings from the microarray data analysis. cd19, cxcl13, fcrl5, and slamf7 serve as diagnostic markers for IPF, providing fresh perspectives regarding the fundamental pathogenesis and molecular mechanisms associated with this condition.

Acetylcholine from tuft cells promotes M2 macrophages polarization in Hirschsprung-associated enterocolitis

BackgroundHirschsprung-associated enterocolitis (HAEC) is one of the most severe complications in patients with Hirschsprung’s disease (HSCR). Previous research has indicated that acetylcholine (ACH) plays an anti-inflammatory role during inflammation by acting on the α7 nicotinic acetylcholine receptor(α7nAchR) to promote the secretion of anti-inflammatory factors. However, the specific role of ACH in HAEC remains unclear. This experiment aims to explore the sources of ACH in HSCR and its anti-inflammatory mechanisms, thereby identifying new directions for the prevention and treatment of HAEC.MethodsWe analyzed single-cell transcriptome data from HSCR to identify cells that secrete ACH and observed their distribution using immunofluorescence. In Ednrb-/- mice, F4/80, iNOS, ARG-1 and CD206 were used to identify and locate M1 and M2 macrophages in different intestinal segments. Western blot, reverse transcription-quantitative polymerase chain reaction, and enzyme-linked immunosorbent assay were used to test the levels of IκBα, tumor necrosis factor-α, interleukin-10, and the macrophage activation pathway proteins JAK2 and STAT3 in different intestinal segments of Ednrb-/- mice. Organoid and cell culture techniques were used to verify the anti-inflammatory mechanism of ACH in vitro models.ResultsscRNA-seq analysis revealed that tuft cells expressed the CHAT protein. In HSCR, aganglionic segments exhibited heightened cholinergic activity compared with dilated ganglionic segments. In HAEC, inflammation was mainly concentrated in the dilated ganglionic segment and was associated with an increase in M1 macrophages, whereas the aganglionic segment showed less inflammation and was associated with an increase in M2 macrophages. Furthermore, in vitro experiments showed that intestinal organoids containing tuft cells promoted an increase in M2 macrophage markers, and ACH promoted M2 macrophage polarization.ConclusionsDifferences in inflammation among various intestinal segments in HAEC may be linked to ACH secreted by tuft cells. Drugs targeting tuft cells have the potential to become important components of HAEC treatment in the future.

Impact of MiRNAs on Wnt-related gene activity in breast cancer

Breast cancer is the most commonly diagnosed cancer in women. The Wnt pathway is involved in the regulation of cell proliferation, differentiation, survival, and migration. Its disruption may promote the induction of breast cancer and its further development. The aim of the study was to identify micro RNA (miRNAs) that could potentially influence the activity of Wnt-related genes in five types of breast cancer in Polish women. Study included patients with five breast cancer subtypes: 130 luminal A, 96 HER2-positive luminal B, 100 HER2-negative luminal B, 36 non-luminal HER2-positive, 43 triple negative breast cancer (TNBC). Tumor tissue was removed during surgery along with a margin of healthy tissue (control group). Expression profile of Wnt-related genes was assessed with mRNA microarrays and reverse transcription quantitative polymerase chain reaction (RT-qPCR). Protein expression was conducted with enzyme-linked immunosorbent assay (ELISA). miRNA profiling was carried out with miRNAs microarrays and the miRDB database. Reduced activity of miR-130a could be related to overexpression of CCND1 and GSK3B. Similarly for miR-199a and GSK3B. High activity of miR-2115 could be associated with downregulation of TCF7L2. WNT5 A overexpression may be linked to low levels of miR-497. In addition, study revealed increased levels of APC, DVL3, LEF1 with reduced activity of FZD4 and TCF7L1 in all five subtypes of breast cancer.

METTL3 Mediates Wnt/β-Catenin Pathway in Epithelial-Mesenchymal Transition of 16HBE Cells Induced by Beryllium Sulphate.

Beryllium (Be) is a recognised environmental toxicant associated with pulmonary fibrosis. Epithelial-mesenchymal transition (EMT), a critical process in cell phenotype conversion, plays a key role in its pathophysiology. Methyltransferase-like 3 (METTL3), a major N6-methyladenosine methyltransferase, regulates gene expression and cellular functions. However, its role in Be-induced EMT remains unclear. In this study, human bronchial epithelial cell line (16HBE cells) were exposed to varying concentrations of beryllium sulphate (BeSO4) to assess changes in METTL3 expression. METTL3 overexpression vectors were constructed, and quantitative reverse transcription-polymerase chain reaction, western blotting and immunofluorescence were used to detect METTL3, EMT markers and Wingless/Integrated (Wnt)/β-catenin pathway proteins. The Wnt/β-catenin pathway inhibitor ICG-001 was also employed to explore the role of the Wnt/β-catenin pathway in BeSO4-induced EMT. The study demonstrated that BeSO4 suppressed METTL3 expression, induced EMT and activated the Wnt/β-catenin pathway in 16HBE cells. Both METTL3 overexpression and ICG-001 pretreatment mitigated BeSO4-induced EMT and Wnt/β-catenin pathway activation. These findings suggest that METTL3 inhibits BeSO4-induced EMT by suppressing the Wnt/β-catenin pathway, offering novel mechanistic insights into beryllium toxicity and a potential therapeutic target for Be-related pulmonary fibrosis.

WFDC3 identified as a prognostic and immune biomarker in pancreatic cancer.

The whey acidic protein four-disulfide core (WFDC) family comprises key modulators of tumor initiation and progression, offering significant potential for diagnostic, prognostic, and therapeutic applications. However, the specific role of WFDCs in the oncogenesis of pancreatic cancer (pancreatic adenocarcinoma [PAAD]) remains incompletely understood. To address this, we conducted an initial investigation using comprehensive bioinformatic analyses to evaluate WFDCs expression patterns across multiple tumor types, with a focus on PAAD. Bulk and single-cell RNA sequencing datasets from the TCGA and GEO repositories were analyzed to assess WFDC3 expression in PAAD tissues. Kaplan-Meier survival analysis was employed to determine the prognostic significance of WFDC3. To explore its biological functions and underlying mechanisms, we performed functional enrichment analyses in combination with immune infiltration assessments. Experimental validation included CCK-8 and EdU proliferation assays, transwell migration and invasion tests, immunofluorescence staining, flow cytometry, LDH release assays, Western blotting, and quantitative reverse transcription PCR. A LASSO regression model was also developed to predict PAAD outcomes. Our findings reveal that WFDCs exhibit context-dependent roles in tumor progression. Specifically, WFDC3 expression was significantly elevated in PAAD and associated with poorer patient prognosis. Functionally, WFDC3 promoted PAAD cell metastasis by inducing epithelial-mesenchymal transition and contributed to immune evasion by suppressing T cell cytotoxicity. In conclusion, our study identifies WFDC3 as a pro-oncogenic factor in PAAD progression, highlighting its potential as both a prognostic biomarker and a therapeutic target for regulating metastasis and immune responses in this malignancy.

Identification of Key Biomarkers of Growth-Related Traits in the Bay Scallop Argopecten irradians irradians via Multi-omics Analysis Strategies.

In an attempt to promote and advance molecular-assisted selective breeding of shellfish, in this study, gills from selected (shell color as the marker trait and growth performance as the target trait) and unselected Argopecten irradians irradians were sampled. 2b-restriction site-associated DNA sequencing, genome-wide association study, comparative transcriptome, comparative small RNA transcriptome, miRNA-mRNA integrated analysis, quantitative real-time reverse transcription-polymerase chain reaction, and weighted gene co-expression network analysis were used to identify candidate SNPs, mRNAs, miRNAs, and miRNA/mRNA pairs with potential selective breeding value at a genomic scale in A. irradians irradians. The results revealed that a total of 6 significant SNPs were correlated closely with at least two examined growth-related traits. In addition, a total of 10 mRNAs and 7 miRNAs were identified to have positive correlations with shell length, shell height, shell width, total weight, and shell color. These candidate mRNAs and miRNAs may form 11 miRNA-mRNA pairs, which have great potential for developing molecular markers for molecular-assisted selective breeding of A. irradians irradians. The findings of this study will benefit the development of molecular-assisted breeding techniques in bay scallop aquaculture.

Immunodynamic axis of fibroblast-driven neutrophil infiltration in acute pancreatitis: NF-κB–HIF-1α–CXCL1

BackgroundAcute pancreatitis (AP) is a sterile inflammation, and 10–20% of cases can progress to severe acute pancreatitis (SAP), which seriously threatens human life and health. Neutrophils and their extracellular traps (NETs) play an important role in the progression of AP. However, the immunodynamic factors between the excessive infiltration of neutrophils during the occurrence of AP have not been fully elucidated.MethodsAdult male C57BL/6 J mice were selected. An AP model was induced by cerulein, and a control group was set up. Single-cell sequencing technology was used to reveal the cell atlas of AP pancreatitis tissue. In vivo, the model mice were treated with anti-Ly6G antibody, DNase I, SC75741, PX-478, and SRT3109 respectively. In vitro, human pancreatic stellate cells were treated with hypoxia, H2O2, NAC, and JSH-2, and co-cultured with neutrophils in Transwell chambers. The severity of inflammation was evaluated, and the molecular mechanism by which fibroblasts exacerbate AP was revealed through techniques such as cell colony formation assay, cell migration assay, cell transfection, immunofluorescence, flow cytometry, Western blot, reverse-transcription quantitative polymerase chain reaction (RT-qPCR), and co-immunoprecipitation (co-IP).ResultsThe study showed that the elimination of neutrophils and NETs could significantly improve AP. Single-cell RNA sequencing (scRNA-seq) indicated that both neutrophils and fibroblasts in pancreatic tissue exhibited heterogeneity during AP. Among them, neutrophils highly expressed CXCR2, and fibroblasts highly expressed CXCL1. Further experimental results demonstrated that the infiltration of neutrophils in the early stage of AP was related to the activation of fibroblasts. The activation of fibroblasts depended on the nuclear factor kappa B (NF-κB) signaling pathway induced by hypoxia. NF-κB enhanced the activation of pancreatic stellate cells (PSCs) and the secretion of CXCL1 by directly promoting the transcription of HIF-1α and indirectly inhibiting PHD2, resulting in the accumulation of HIF-1α protein. The NF-κB–HIF-1α signal promoted the secretion of CXCL1 by fibroblasts through glycolysis and induced the infiltration of neutrophils. Finally, blocking the NF-κB–HIF-1α–CXCL1 signaling axis in vivo reduced the infiltration of neutrophils and improved AP.ConclusionsThis study, for the first time, demonstrated that activation of fibroblasts is one of the immunological driving factors for neutrophil infiltration and elucidated that glycolysis driven by the NF-κB–HIF-1α pathway is the intrinsic molecular mechanism by which fibroblasts secrete CXCL1 to chemotactically attract neutrophils. This finding provides a highly promising target for the treatment of AP.

Influences of Magnesium Isoglycyrrhizinate on the Pharmacokinetics of Almonertinib in Rats and Its Potential Mechanisms.

Almonertinib has been approved for the first-line therapy of advanced epidermal growth factor receptor (EGFR) mutation-positive non-small cell lung cancer (NSCLC). As a hepatoprotective agent, magnesium isoglycyrrhizinate (MgIG) was widely used in the treatment of drug-induced liver injury. Although Almonertinib is commonly prescribed in combination with MgIG in clinical application, the interaction research between them has not been reported. This research was conducted to explore the influences of MgIG on the pharmacokinetics of Almonertinib in rats and to investigate its possible molecular mechanisms. The plasma concentration of Almonertinib in male Sprague-Dawley (SD) rats was determined by ultra-performance liquid chromatography-tandem mass spectrometer (UPLC-MS/MS), the messenger RNA (mRNA) and protein expression levels of CYP3A1 (homologous to human CYP3A4) and P-glycoprotein (P-gp) in rat livers were measured by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blot analyses. MgIG increased systemic exposure of Almonertinib significantly, resulting in increased AUC0-t, AUC0-∞, Cmax, and t1/2, and decreased CLz. Furthermore, MgIG can markedly down-regulate the expression levels of CYP3A1 and P-gp in rat livers. There is a drug-drug interaction (DDI) between MgIG and almonertinib, which leads to an increase of systemic exposure for almonertinib. Meanwhile, CYP3A1 and P-gp in the liver may be the primary targets of mediating the DDI, but further clinical trials are needed to confirm these results.

To determine the role of TRIT1 in the diagnosis, prognosis and immunoinvasion of liver hepatocellular carcinoma

BackgroundTRIT1 is identified as a potential tumor suppressor gene that may be involved in tumor development. Existing research indicates that TRIT1 is significant in the development of certain cancers. However, its specific role in liver cancer remains elusive.MethodsExpression profiles and clinical data of liver hepatocellular carcinoma (LIHC) patients were retrieved from The Cancer Genome Atlas (TCGA) database. The TRIT1 gene levels between LIHC tissues and normal tissues were compared using the Wilcoxon rank-sum test. Additionally, TRIT1 expression levels were further examined via reverse transcription quantitative polymerase chain reaction (RT-qPCR). Functional enrichment analysis was performed to elucidate the biological pathways associated with TRIT1. Immune cell infiltration patterns were evaluated using single-sample gene set enrichment analysis (ssGSEA). The methylation status of the TRIT1 gene were analyzed using the UALCAN and MethSurv databases. Cox regression analysis and Kaplan-Meier (KM) methods were employed to determine the prognostic value of TRIT1. To create a practical tool for predicting overall survival over time, a nomogram was constructed.ResultsThe analysis revealed that TRIT1 expression is significantly higher in LIHC tissues compared to normal tissues. Furthermore, elevated TRIT1 levels were found to be associated with specific subtypes of LIHC, including T3 and stage III. Importantly, TRIT1 overexpression was identified as a negative prognostic marker for overall survival in LIHC patients. Additionally, hypermethylation of the TRIT1 gene was associated with poor prognosis. Moreover, this study demonstrated that high TRIT1 levels were correlated with reduced levels of cytotoxic immune cells in the tumor microenvironment, including B cells, cytotoxic cells, and plasmacytoid dendritic cells (pDCs).ConclusionsThis study provides the first evidence that the presence of TRIT1 can serve as a reliable marker for diagnosis and prognostication of hepatocellular carcinoma. Moreover, TRIT1 emerges as a critical indicator of the potential for cancer infiltration and invasion of the immune system, holding significant implications for the development of targeted therapies for hepatocellular carcinoma.

Association of miR-149 gene rs2292832 polymorphism with necrotizing enterocolitis in preterm infants

Background. Necrotizing enterocolitis (NEC) is a prevalent and challenging intestinal disease in premature infants, lacking a specific pathogen consistently associated with its occurrence. Effectively preventing and treating NEC to reduce mortality rates remains a significant contemporary challenge. The present study aimed to explore the correlation between microRNA-149 gene polymorphism and NEC in premature infants in a Chinese Han population. Methods. The expression levels of serum miR-149 were determined using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Polymorphism detection of the miR-149 gene rs2292832 polymorphism was performed by polymerase chain reaction. Multivariate logistic regression analysis was employed to investigate the association between the rs2292832 polymorphism and risk factors for NEC in preterm infants. Results. General clinical data were compared between 102 preterm infants diagnosed with NEC and 263 preterm infants without NEC. Significant differences were observed in gestational age and birth weight. However, no significant differences were found in antenatal steroid use, sex, or feeding patterns between the two groups. The expression level of serum miR-149 was significantly reduced in premature infants with NEC, and there were differences in the allele frequency of the miR-149 rs2292832 polymorphism between the NEC group and control group. Specifically, the T allele and TT genotype of rs2292832 were associated with an increased susceptibility to NEC. Furthermore, both gestational age and the rs2292832 polymorphism showed a significant association with NEC risk, with the rs2292832 polymorphism of miR-149 being identified as the most prominent risk factor for NEC development in preterm infants. Conclusions. The rs2292832 gene polymorphism of miR-149 may potentially exert an influence on susceptibility to NEC.

Exploring the role of miRNAs in Hepatocellular Carcinoma: Insights into signaling pathways and their regulatory mechanisms

Objective: This prospective case-control study aimed to assess the predictive value of the expression levels of Hsa-miR-200-3p, Hsa-miR-155-5p, Hsa-miR-20a-5p, Hsa-miR-221-3p, Hsa-miR-34a-5p, Hsa-let-7b-5p, and Hsa-miR-125b-5p microRNAs (miRNAs) in the diagnosis, prognosis, and treatment response of hepatocellular carcinoma (HCC) patients. The study includes 20 healthy controls and 25 HCC patients. Methods: The study was designed as a prospective case-control study and was conducted between 2023 to 2024 at Ataturk University, in collaboration with the Departments of Organ Transplantation, Medical Biology and Medical Pathology. miRNA expression levels were examined in HCC tissue samples. RNA was extracted from paraffin-embedded tissues, and miRNA expression levels were measured using reverse transcription-quantitative PCR. The relationships between clinical factors and miRNA expression levels were assessed. Results: Significant differences in expression levels were found between HCC tissues and healthy control tissues for Hsa-miR-20a-5p, Hsa-miR-125b-5p, and Hsa-let-7b-5p. A significant correlation was observed between Hsa-miR-34a-5p and γ-glutamyl transferase levels (P&lt;0.05), as well as between Hsa-miR-20a-5p and albumin levels (P&lt;0.05). Significant differences in expression levels were found between patients with and without HBV infection for let-7b-5p (P=0.007) and miR-200-3p (P=0.013). Conclusion: This study demonstrates that miRNAs, particularly Hsa-miR-20a-5p and Hsa-miR-34a-5p, may regulate the Wnt/β-catenin pathway and influence the progression of HCC. Disease progression was assessed by comparing miRNA expression levels with clinical markers (such as γ-glutamyl transferase and albumin). High expression of Hsa-miR-20a-5p affects the Wnt/β-catenin pathway and plays a significant role in disease processes. Additionally, the correlation between Hsa-miR-34a-5p and γ-glutamyl transferase indicates disease progression. However, further advanced studies are required to better understand how the Wnt/β-catenin pathway is regulated by miRNAs and the impact of this regulation on the disease. doi: https://doi.org/10.12669/pjms.41.5.11444 How to cite this: Altundas N, Balkan E, Kizilkaya M, Demirci E. Exploring the role of miRNAs in Hepatocellular Carcinoma: Insights into signaling pathways and their regulatory mechanisms. Pak J Med Sci. 2025;41(5):1441-1446. doi: https://doi.org/10.12669/pjms.41.5.11444 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Induced pluripotent stem cell-derived mesenchymal stem cells (iMSCs) inhibit M1 macrophage polarization and reduce alveolar bone loss associated with periodontitis

BackgroundPeriodontitis is a chronic inflammatory disease and macrophages play a pivotal role in the progression of periodontitis. Mesenchymal stem cells (MSCs) have emerged as potential therapeutic agents for the treatment of periodontitis due to their immunomodulatory properties and capacity for tissue regeneration. Compared to conventionally derived MSCs, induced pluripotent stem cell-derived MSCs (iMSCs) offer distinct advantages as promising candidates for MSC-based therapies, owing to their non-invasive acquisition methods and virtually unlimited availability. This study aims to investigate the effects and mechanisms of iMSCs in modulating macrophage polarization and alleviating periodontitis-related alveolar bone loss.MethodsiMSCs were generated from iPSCs and characterized for differentiation potential. The effects of iMSCs on macrophage polarization were evaluated using THP-1-derived macrophages under inflammatory conditions (LPS and IFN-γ stimulation). Co-culture assays, cytokine analysis, reactive oxygen species (ROS) detection, transcriptomic analysis, flow cytometry, reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and western blot analysis were performed to elucidate the underlying mechanisms. The therapeutic potential of iMSCs was assessed in a ligature-induced periodontitis mouse model using micro-CT, histological analysis, and immunofluorescence staining.ResultsiMSCs inhibit M1 macrophage polarization through the suppression of the NF-κB signaling pathway. Additionally, iMSCs reduce the production of pro-inflammatory cytokines (IL-1β, IL-17) and reactive oxygen species (ROS), while enhancing the secretion of anti-inflammatory cytokines (IL-10) and growth factors (VEGF), thereby improving the inflammatory microenvironment. Under inflammatory conditions, iMSCs preserve the osteogenic potential of periodontal ligament stem cells (PDLSCs) and alleviate alveolar bone loss in mice with periodontitis. In vivo, iMSCs reduce the number of M1 macrophages and inhibit the activation of NF-κB in periodontal tissues, supporting their anti-inflammatory and immunomodulatory effects.ConclusioniMSCs demonstrate significant therapeutic potential in periodontitis by modulating macrophage polarization, reducing oxidative stress, and mitigating alveolar bone loss associated with the disease. These findings provide new insights into the mechanisms of iMSCs and their application as cell-based therapies for periodontal diseases.

Identification and expression analysis of PAL genes related to chlorogenic acid synthesis in Vaccinium dunalianum Wight

Phenylalanine ammonia-lyase (PAL) is the first key enzyme in the metabolic pathway of phenylpropanoid. Chlorogenic acid (CGA) is an important secondary metabolite in the phenylpropanoid metabolic pathway of plants. However, the role of the PAL gene in CGA biosynthesis in Vaccinium dunalianum Wight is still unclear. In this study, seven PAL genes (VdPAL1–VdPAL7) were cloned and analyzed through full-length transcriptome sequencing combined with reverse transcription polymerase chain reaction (RT-PCR) amplification in V. dunalianum. Bioinformatics analysis revealed that the seven VdPAL genes contain complete open reading frames (1,860–2,148 bp) encoding hydrophilic proteins of 619–715 amino acids, with molecular weights ranging from 67.44 to 77.70 kDa. The VdPAL proteins contain the PLN02457 domain, with the secondary structures predominantly composed of α-helices and the tertiary structures exhibiting a characteristic “hippocampus” conformation. Phylogenetic analysis grouped these VdPAL genes into three subfamilies: VdPAL1, VdPAL3, and the VdPAL2/4/5/6/7 cluster. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis showed that the expression levels of the VdPAL1 and VdPAL4/5/6/7 genes were significantly higher in both tender and mature leaves, in flower buds, flowers, green and red fruits, and in green and red fruit stems than those of VdPAL2 and VdPAL3. In the same tissue type, except for VdPAL2, the expression levels of the other six PAL genes were significantly higher in young tissues compared with mature tissues. The PAL gene family mediates the biosynthesis of CGA in V. dunalianum, with the combined expression of VdPAL1/2/4/5/6/7 showing a positive correlation with the CGA content across the analyzed tissues. These results provide experimental evidence for understanding the composition, structural characteristics, and biological functions of the PAL genes in V. dunalianum.

Transcriptions of ACO and ACS genes are involved in nitrate-dependent root growth of maize seedlings

Improving nitrogen use efficiency (NUE) is one of the major objectives for crop breeding. As nitrate signaling plays pivotal roles in nitrogen use of plants, factors in this pathway might be valuable for improving the NUE of maize. In this research, we performed Gene Set Enrichment Analysis (GSEA) of maize transcriptomes in response to nitrate and found that the ethylene action pathway might participate in nitrate signaling. Through a modified reciprocal best hit approach, we obtained 16 maize aminocyclopropane-1-carboxylic acid (ACC) oxidase (ACO) and four ACC synthase (ACS) homologs in maize genome. In silico analyses and the reverse transcription quantitative PCR assays demonstrated that ZmACCO7, ZmACCO5, ZmACCO15, ZmACCO35, and ZmACCO31 are the top five highly expressed ACO genes, and ZmACS1 is the most highly expressed ACS gene in the primary and seminal roots of maize. We discovered that ACO and ACS genes have different regulatory modes in response to nitrate provision. Some ACO genes, which are mainly expressed in root regions far from the root tip like ZmACCO7, are repressed by nitrate, while the others, which are mainly expressed in root regions near the root tip like ZmACCO5, are induced by nitrate. ZmACS1, which has more uniform expression across maize roots, is induced in root regions near the root tip and repressed in regions far from the root tip. A phenotypic analysis indicated that upregulation of ACO and ACS genes by nitrate is linked to repression of axial root elongation by nitrate while the downregulation of these genes is associated with the promotion of growth of lateral roots of the axial roots. In addition, differences in regulation of ACO and ACS genes by nitrate were observed between genotypes, which is related to the differences in the responses of their primary root growth to nitrate. These results suggested that the ethylene synthesis pathway is involved in the responses of maize roots to nitrate, which is associated with the remodeling of maize root architecture by nitrate.

Salinity-induced virulence alteration of Aeromonas hydrophila isolated from Scatophagus argus: insights from transcriptomic profiling and phenotypic characterization

BackgroundThe emerging foodborne pathogen, Aeromonas hydrophila, co-infects humans and animals, especially fish, threatening aquacultural production and public health. Previously, we found that Scatophagus argus, a widely cultivated fish species with high economic value, exhibited enhanced growth but increased susceptibility to A. hydrophila infection under freshwater conditions compared to seawater conditions. However, the exact mechanisms involved remain unclear.ResultsOur study demonstrated that the enhanced virulence of A. hydrophila 201416, isolated from S. argus, in response to increasing salinity was associated with altered quorum sensing-related gene expression and regulated behaviors. Results from virulence assays incorporating phenotypic characterization indicated that elevated salinity levels (from 0 to 35‰) significantly hindered Ah201416 infection of S. argus. This trend correlated with increased biofilm mass and swimming motility, yet was inversely related to bacterial growth. RNA-sequencing and quantitative reverse transcriptional PCR analysis confirmed significant upregulation of genes related to flagellar assembly (flgB, flgH, flgC, flgI, flhA, and fliA), bacterial secretion (HlyD and Ahh1), and quorum sensing (AhyR, LuxO, and LuxE) of Ah201416 in response to elevated salinity. These findings suggested that increased salinity not only enhanced the virulence of Ah201416 but also bolstered the resistance of S. argus, thereby mitigating its susceptibility.ConclusionsThis study provides deeper insights into the microbial risks associated with A. hydrophila in aquacultural production, which is critical to developing effective prevention and control strategies and ensuring a safe seafood supply.Clinical trial numberNot applicable.