Quantitative RT-PCR Analysis Research Articles

Dexmedetomidine Blocks NCOA4-Dependent Ferritinophagy to Confer Ferroptosis Resistance in Lung Ischemia Reperfusion Injury via Targeting NRF2.

Lung ischemia reperfusion injury (LIRI) represents an evitable but significant pathologic complication post pulmonary transplantation. Dexmedetomidine (Dex) that is extensively applied as an anesthetic adjuvant in the intensive care setting has increasingly presented outstandingly protective effect on LIRI. This article concerns the elaborate role of Dex in ferroptosis after LIRI and the correlative downstream mechanism. Upon hypoxia/reoxygenation (H/R) in human (A549) and mouse (MLE-12) alveolar epithelial cells, reverse transcription-quantitative PCR and western blot analysis tested nuclear receptor coactivator 4 (NCOA4) expression. CCK-8 kit determined cell viability. Western blot analysis and immunofluorescence assay estimated ferritinophagy. C11-BODIPY 581/591 staining, western blot analysis, assay kits and ferro-orange staining appraised ferroptosis. Molecular docking technology investigated the binding affinity between Dex and nuclear factor erythroid 2-related factor 2 (NRF2). Cell viability was eliminated and ferritinophagy was aggravated in A549 and MLE-12 cells in response to H/R. Disturbance of NCOA4 or treatment with Dex suppressed the ferroptosis in H/R-stimulated cells. Also, Dex docked with NRF2 and upregulated NRF2 to concentration-dependently obstruct NCOA4-mediated ferritinophagy and ferroptosis in H/R-challenged cells. Collectively, Dex might protect against NCOA4-mediated ferritinophagy through targeting NRF2, thereby alleviating ferroptosis during LIRI.

Maize ZmWRKY71 gene positively regulates drought tolerance through reactive oxygen species homeostasis.

Drought stress severely affects plant growth and yield. The plant-specific WRKY transcription factors play an important role in regulating the plant response to abiotic stresses. In this study, we identified a group I WRKY gene from maize, designated ZmWRKY71. Real-time quantitative reverse transcription-PCR analysis revealed that ZmWRKY71 was predominantly expressed in the roots and was induced by drought. ZmWRKY71 was localized in the nucleus and showed transcriptional activity in yeast. Heterologous overexpression of ZmWRKY71 improved drought tolerance in yeast and Arabidopsis. Compared with the wild type, the overexpression lines showed a higher survival rate under drought stress with reduced malondialdehyde content and elevated antioxidant enzyme activities. In contrast, mutation of ZmWRKY71 in maize leads to increased sensitivity to drought stress, reduced survival, elevated concentrations of reactive oxygen species, and increased malondialdehyde content. RNA-sequencing analysis revealed that the expression patterns of genes associated with translation, membrane, and oxidoreductase activity pathways were altered under drought stress. Yeast one-hybrid, dual-luciferase, and electrophoretic mobility shift assays confirmed that ZmWRKY71 was capable of directly binding to the W-box element in the promoter region of ZmPOD42 (Zm00001eb330550). Taken together, the results show that ZmWRKY71 positively regulates maize drought tolerance. This research enriches the drought tolerance gene pool for maize and provides a theoretical basis for maize drought tolerance breeding.

Integration of Single-Cell and Bulk Transcriptomes to Identify a Poor Prognostic Tumor Subgroup to Predict the Prognosis of Patients with Early-stage Lung Adenocarcinoma.

Background: Single-cell RNA sequencing (scRNA-seq) has emerged as a pivotal technology for investigating novel therapeutic targets in cancer. Despite its significance, there remains a scarcity of studies utilizing this technology to address treatment strategies specifically tailored for early-stage lung adenocarcinoma (LUAD). Consequently, this study aimed to investigate the tumor microenvironment (TME) characteristics and develop a prognostic model for early-stage LUAD. Methods: The markers identifying cell types were obtained from the CellMarker database and published research. The SCEVAN package was employed for identifying malignant lung epithelial cells. Single-cell downstream analyses were conducted using the SCP package, encompassing gene set enrichment analysis, enrichment analysis, pseudotime trajectory analysis, and differential expression analysis. Calibration curves, receiver operating characteristic curves, and decision curve analysis were employed to assess the performance of the prognostic model for LUAD. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR), western blot, cell transfection, cell proliferation, and cell invasion assays were performed to validate the expression and biological function. Results: Seven cell types were distinguished in the scRNA-seq dataset through the utilization of cell markers documented in published literature. Four subpopulations of early-stage LUAD tumor cells exhibited a high degree of heterogeneity. The prognostic model constructed by PERP and KRT8 showed a great prediction for distinguishing the early-stage LUAD and normal tissues. The validation of PERP and KRT8 expression levels was carried out through both RT-qPCR and western blot analyses. Eventually, in vitro experiments, including CCK8, colony formation, EdU, and transwell assays, confirmed that KRT8 and PERP could promote LUAD cell proliferation and migration. Conclusions: Our study provided a comprehensive characterization of the TME in LUAD through integrative single-cell and bulk transcriptomic analyses. We identified dynamic transitions from normal epithelial cells to tumor cells, revealing the heterogeneity and evolution of malignant LUAD cells. The novel prognostic model based on KRT8 and PERP demonstrated robust predictive performance, offering a promising tool for early-stage LUAD risk stratification. Functional experiments further confirmed that KRT8 and PERP promote tumor proliferation and migration, providing new insights into their roles as therapeutic targets.

Liraglutide mitigates dexamethasone-induced fatty acid synthase (FASN) and the cluster of differentiation36 (CD36) expression: a potential treatment for glucocorticoid-induced non-alcoholic fatty liver disease (NAFLD).

The clinical use of dexamethasone (DXM) is associated with the development of non-alcoholic fatty liver disease (NAFLD). However, the mechanisms by which DXM-induced NAFLD is still incompletely known. Therefore, the current study aims to test the hypothesis that DXM-induced NAFLD is mediated by dysregulation of key genes involved in lipid metabolism and liraglutide (LG) can ameliorate these effects. The histopathological and biochemical analysis assessed the effects of DXM and/or LG in liver tissue. The computational analysis was performed to detect the glucocorticoid response elements (GRE) in the promotor regions of FASN and CD36 genes. The effects of DXM and LG on the expression of FASN and CD36 were determined by real-time quantitative reverse transcription PCR (RT-qPCR), western blot (WB) and immunohistochemical (IHC) analyses. The NAFLD induced by high-fat diet (HFD) and DXM was manifested by increased levels of liver enzymes, deterioration of histological architecture of the liver tissue and accumulation of fat droplets. Computational analysis revealed that the promotor regions of FASN and CD36 harper several GRE. Most importantly, treatment with DXM decreased phosphorylated adenosine monophosphate-activated protein kinase (p-AMPK) levels, while LG upregulate it. In addition, treatment with DXM increased expression of FASN and CD36, whereas LG ameliorated these effects in a dose-dependent manner. DXM-induced NAFLD is mediated by upregulation of FASN and CD36 expression which may be attributed to GRE. LG coupling with DXM mitigates this induction by downregulating FASN and CD36 levels and therefore mitigates the development of NAFLD.

Edaravone Improves Motor Dysfunction Following Brachial Plexus Avulsion Injury in Rats.

Brachial plexus root avulsion (BPRA) is often caused by road collisions, leading to total loss of motor function in the upper limb. At present, effective treatment options remain limited. Edaravone (EDA), a substance that eliminates free radicals, exhibits numerous biological properties, including neuroprotective, antioxidant and anti-inflammatory effects. However, the specific role and molecular mechanisms of EDA in the treatment of BPRA remain to be fully elucidated. The present study used a rat model of BPRA, following avulsion of the fifth, sixth and seventh cervical (C5, C6 and C7) anterior roots. Notably, C6 was replanted following a subcutaneous injection of either saline or 30 mg/kg/day EDA for seven continuous days. Subsequently, behavioral, histochemical, Western blot and reverse transcription-quantitative PCR (RT-PCR) analyses were conducted. Results of the present study revealed that treatment with EDA improves motor dysfunction, indicated by the increased Grooming test score, usage of the affected limb, and Irvine, Beatties and Bresnahan (IBB) score, following BPRA. In addition, EDA reduced the death of motoneurons (MNs), indicated by the increased number of Nissl-positive neuron, at the site of the affected limb, inhibited neuroinflammation and cellular pyroptosis, indicated by the decreased expression levels of IL-1β, IL-6, TNF-α, IL-18, p-p65, NLRP3, GSDMD and Caspase-1, improved the morphology of the abnormal myocutaneous nerve fibers, promoted axon remyelination, indicated by increased mRNA expression levels of remyelination-associated genes, including egr2, GAP-43, hmgcr, L1CAM, mpz, pmp22 and prx and demyelination-associated genes, including ngfr, notch1, pou3f1 and sox2, and alleviated muscle atrophy, indicated by the increased weight and volume of biceps brachii muscle, and the decreased number of fibroblasts and increased diameters in the fibers. Collectively, results of the present study suggested that EDA may support axonal remyelination and inhibit pyroptosis-associated neuroinflammation, enhancing MN survival and facilitating functional motor recovery. Thus, the present study may provide a novel theoretical basis for the use of EDA in the treatment of BPRA.

Identification and characterisation of temporal abundance of microRNAs in synovial fluid from an experimental equine model of osteoarthritis.

MicroRNAs, a class of small noncoding RNAs, serve as post-transcriptional regulators of gene expression and are present in a stable and quantifiable form in biological fluids. MicroRNAs may influence intra-articular responses and the course of disease, but very little is known about their temporal changes in osteoarthritis. To identify miRNAs and characterise the temporal changes in their abundance in SF from horses with experimentally induced osteoarthritis. We hypothesised that the abundance of miRNA would change during disease progression. In vivo experiments. RNA extracted from synovial fluid obtained sequentially (Day 0, 28 and 70) from nine horses with experimentally induced osteoarthritis was subjected to small RNA sequencing using the Illumina Hiseq 4000 sequencing platform. Differentially abundant miRNAs underwent further validation and mapping of temporal abundance (Day 0, 14, 17, 21, 28, 35, 42, 49, 56, 63 and 70 days after osteoarthritis induction) by microfluidic reverse transcription quantitative real-time PCR. Bioinformatic analyses were performed to predict potential biological associations and target genes of the differentially abundant microRNAs. Small RNA sequencing revealed 61 differentially abundant microRNAs at an early osteoarthritis stage (Day 28), and subsequent reverse transcription quantitative real-time PCR analysis validated 20 of these. Significant biological functions of the differentially abundant microRNAs were apoptosis, necrosis, cell proliferation and cell invasion. Following validation, four microRNAs (miRNA-199b-3p, miRNA-139-5p, miRNA-1839 and miRNA-151-5p) were detected in more than 50% of the synovial fluid samples and had higher abundance in osteoarthritic than in control joints. Limited sample size. This is the first study to determine longitudinal changes in synovial fluid microRNA abundance in an equine model of osteoarthritis. Larger studies are needed in naturally occurring osteoarthritis to interrogate putative changes identified by this study.

Evaluation of Salivary and Serum micro RNA 146a, 200c and its Target Gene PTEN in Chronic Periodontitis Patients and their Response to Non-Surgical Periodontal Therapy.

Periodontitis destroys the tooth's supporting structures and attachment apparatus. Local or systemic factors can cause it. Traditionally, diagnosis is based on clinical parameters that may not consistently reflect an accurate confirmation. Biochemical and genetic analyses can provide deeper insights. MicroRNAs (miRNAs) regulate the immune and inflam-matory response to microbial pathogens. Detecting and evaluating miRNAs can be an important diagnostic parameter. This study aimed to assess the expression of miRNA 146a,200c, and its target gene PTEN to non-surgical periodontal therapy in serum and saliva. This interventional comparative study was conducted on 120 patients of both genders, ages between 35 and 55. Non-surgical periodontal therapy (NSPT) scaling and root planing were performed on all subjects, and their saliva and serum samples were collected before and after 8 weeks of NSPT. Quantitative rt-PCR (reverse transcriptase Polymerase Chain Reaction) analysis was conducted on all samples. The statistical analysis was done using SPSS version 22, and comparisons were made using paired t-tests, independent t-tests, and Pearson's correlation coefficient. The statistical significance level was set at a 'P' value of less than 0.05. It has been observed that there was a significant difference of miRNA in both serum and saliva samples 146a,200c, and the PTEN gene expression, from the beginning to 8 weeks. Sig-nificant variation was not observed when comparing the levels between serum and saliva. miRNA 146A, 200c, and PTEN genes are interrelated with periodontitis. We can consider them as future biomarkers of periodontal diseases.

Efficacy of metformin and verteporfin treatment alone or in combination in a murine head and neck cancer xenograft model.

Despite treatment advances, head and neck squamous cell carcinoma (HNSCC) still poses a significant global health challenge. Combination therapies have emerged as more effective strategies than traditional chemotherapy in clinical practice by improving tumor response rates and patient survival while minimizing treatment-related toxicity. This study investigates the anticancer effects of metformin and verteporfin (Yes-associated protein 1 [YAP1] inhibitor) alone or in combination in HNSCC using vitro and in vivo approaches. Quantitative RT-PCR analysis of HNSCC cell lines reveals upregulation of YAP1 and related genes in the Hippo signaling pathway. Cell viability assays demonstrate a beneficial synergistic effect between metformin and verteporfin in inhibiting HNSCC tumor growth. In male BALB/cAJcl-nu/nu mice harboring HNSCC tumor xenografts, intraperitoneal administration of metformin and verteporfin enhances the inhibitory effect on tumor growth and suppresses YAP1 nuclear translocation when compared to vehicle or monotherapies. Furthermore, combination of these drugs reduces tumor cell proliferation (marked by Ki-67) and inhibits phosphoS6 ribosomal protein, as observed through immunofluorescent and immunohistochemical (IHC) analyses. The in vivo study underscores the therapeutic potential of the dual targeting approach with metformin and verteporfin in treating HNSCC, with minimal toxicity.

Detection of cervical precancerous lesions and cancer by small-scale RT-qPCR analysis of oppositely deregulated mRNAs pairs in cytological smears.

Cervical screening, aimed at detecting precancerous lesions and preventing cancer, is based on cytology and HPV testing. Both methods have limitations, the main ones being the variable diagnostic sensitivity of cytology and the moderate specificity of HPV testing. Various molecular biomarkers are proposed in recent years to improve cervical cancer management, including a number of mRNAs encoded by human genes involved in carcinogenesis. Many scientific papers have shown that the expression patterns of cellular mRNAs reflect the severity of the lesion, and their analysis in cervical smears may outperform HPV testing in terms of diagnostic specificity. However, such analysis has not yet been implemented in broad clinical practice. Our aim was to devise an assay detecting severe cervical lesions (≥HSIL) via analysis of cellular mRNA expression in cytological smears. Through logistic regression analysis of a reverse-transcription quantitative PCR (RT-qPCR) dataset generated from analysis of six mRNAs in 167 cervical smears with various cytological diagnoses, we generated a family of linear classifiers based on paired mRNA concentration ratios. Each classifier outputs a dimensionless decision function (DF) value that increases with lesion severity. Additionally, in the same specimens, the HPV genotyping, viral load assessment, diagnosis of cervicovaginal microbiome imbalance and profiling of some relevant mRNAs and miRNAs were performed by qPCR-based methods. The best classifiers were obtained with pairs of mRNAs whose expression changes in opposite directions during lesion progression. With this approach based on a five-mRNA combination (CDKN2A, MAL, TMPRSS4, CRNN, and ECM1), we generated a classifier having ROC AUC 0.935, diagnostic sensitivity 89.7%, and specificity 87.6% for ≥HSIL detection. Based on this classifier, a two-tube RT-qPCR based assay was developed and it confirmed the preliminary characteristics on 120 cervical smears from the test sample. DF values weakly correlated with HPV loads and cervicovaginal microbiome imbalance, thus being independent markers of ≥HSIL risk. Thus, we propose a high-throughput method for detecting ≥HSIL cervical lesions by RT-qPCR analysis of several cellular mRNAs. The method is suitable for the analysis of cervical cytological smears prepared by a routine method. Further clinical validation is necessary to clarify its clinical potential.

Modification of Gene Expression Involved in Alkaloid Production in Opium Poppy by VIGS Combined With Pretreatment of Macerozyme Enzyme.

Papaver somniferum L., a medicinal plant renowned for its pharmaceutical alkaloids, has captivated scientific interest due to its rich secondary metabolite profile. This study explores a novel approach to manipulating alkaloid biosynthesis pathways by integrating virus-induced gene silencing (VIGS) with macerozyme enzyme pretreatment. Targeting key genes in the benzylisoquinoline alkaloid (BIA) pathway (CODM, T6ODM, COR, DIOX2), the research aimed to elucidate the transformative potential of enzymatic preconditioning in somatic embryo cultures. To address the cell wall barrier, a known limitation in genetic manipulation, macerozyme pretreatment was employed, significantly enhancing gene silencing efficacy. Quantitative reverse transcription PCR analyses revealed significant alterations in gene expression profiles with macerozyme pretreatment, whereas no changes were observed in its absence. The T6ODM + DIOX combination was the most effective, reducing CODM, T6ODM, and DIOX2 expression by 72%, 65%, and 60%, respectively. Conversely, T6ODM expression increased by up to 107% in the CODM treatment. Notably, COR expression displayed dual regulatory dynamics, with suppression (47% decrease in T6ODM + DIOX) and enhancement (49% increase in CODM+DIOX) observed under different conditions. These findings underscore the complex interplay of gene regulation in the morphine biosynthesis pathway. This study highlights the critical role of macerozyme enzymatic pretreatment in overcoming cell wall barriers, enabling effective VIGS applications in somatic suspension cultures. The combination of VIGS and enzymatic pretreatment provides a robust platform for targeted metabolic engineering, offering insights into the regulation of morphine biosynthesis and paving the way for advancements in pharmaceutical alkaloid production and functional genomics in medicinal plants.

RS24090, a TetR family transcriptional repressor, negatively affects the rimocidin biosynthesis in Streptomyces rimosus M527

The TetR family of regulators (TFRs), commonly reported as repressors, plays a role in regulating secondary metabolite production in Streptomyces. In this study, we sought to elucidate the relationship between TFRs and rimocidin production of Streptomyces rimosus M527. Through transcriptomic analysis, we identified the protein RS24090, which exhibited significant differential expression. Phylogenetic analysis of its amino acid sequence and structural alignment predicted it to be a TetR family regulator. Thus, RS24090 was named TetR24. The role of TetR24 in biosynthesis of rimocidin was verified through gene-deletion, −complementation, and -overexpression experiments. The TetR24 gene-deletion mutant (ΔTetR24), which was generated using CRISPR/Cas9 technology, produced 38.08 % more rimocidin than the wild-type (WT) strain M527. Complementary expression of the TetR24 gene in the mutant ΔTetR24 restored rimocidin production to levels comparable to the WT strain. In contrast, the recombinant strain M527-TetR24, which harbored an overexpression of the TetR24 gene, exhibited a 40.31 % decrease in rimocidin production compared to the WT strain. A similar trend in the transcription levels of the rim genes (rimA, rimC, rimG, rimR1, and rimR2), all located in the rimocidin biosynthetic gene cluster, was revealed by quantitative RT-PCR analysis in M527-ΔTetR24, M527-ΔTetR24::TetR24, and M527-TetR24. EMSA and DNase I footprinting assays confirmed that TetR24 regulates the transcription of rim genes by binding to promoter regions of rimA and rimR2.

MicroRNA-221 protects myocardial contractility in myocardial ischemia/reperfusion injury through phospholamban.

To investigate the effects and mechanisms of miRNA 221 on myocardial ischemia/reperfusion injury (MIRI) in mice through the regulation of phospholamban (PLB) expression. The MIRI mouse model was created and mice were divided into sham, MIRI, MIRI+ 221, and MIRI+ scr groups, with miRNA 221 overexpression induced in the myocardium of MIRI mice by targeted myocardial injection. Quantitative RT-PCR analysis was performed to observe the variation in miRNA 221, PLB, SERCA2, RYR2, NCX1, Cyt C and caspase 3 mRNA levels in myocardium, while Western blot assessed the levels of PLB, p-PLB (Ser16), p-PLB (Thr17), SERCA2, RYR2, NCX1, Cyt C and caspase 3 proteins. Changes in the structural integrity of the mouse heart were identified with HE and MASSON staining, while TUNEL staining was used to evaluate the TUNEL-positive cells of cardiomyocytes. Changes in myocardium calcium concentration were detected with reagent kits and the targeting interaction between miRNA 221 and PLB was evaluated using a luciferase reporter assay. In the myocardium of MIRI mice, miRNA 221 level was significantly reduced, while the levels of PLB, p-PLB (Ser16), p-PLB (Thr17), and apoptosis-related genes caspase 3, and Cyt C were increased markedly, as well as calcium levels in myocardium. Following the overexpression of miRNA 221 in myocardium, there was a marked alleviation of myocardial injury and cardiomyocyte apoptosis and necrosis, significant enhancement of left ventricular systolic function, and marked decrease in the levels of PLB, p-PLB (Ser16), p-PLB (Thr17), caspase 3 and Cyt C, as well as a significant decrease in total calcium levels in myocardium. miRNA 221 can alleviate myocardial injury in mouse myocardial ischemia/reperfusion by suppressing the expression of PLB, thus reducing calcium overload in myocardium.

Interaction Between YTH Domain-Containing Family Protein 2 and SET Domain-Containing Lysine Methyltransferase 7 Suppresses Autophagy in Osteoarthritis Chondrocytes, Exacerbating Cartilage Damage.

Osteoarthritis (OA) is characterized by progressive cartilage degeneration mediated by various molecular pathways, including inflammatory and autophagic processes. SET domain-containing lysine methyltransferase 7 (SETD7), a methyltransferase, has been implicated in OA pathology. This study investigates the expression pattern of SETD7 in OA and its role in promoting interleukin-1 beta (IL-1β)-induced chondrocyte injury through modulation of autophagy and inflammation. The expression of SETD7 in cartilage tissues from OA patients and healthy controls was quantified using quantitative reverse transcription PCR and Western blot analysis. Small interfering RNA targeting SETD7 (si-SETD7) was transfected into human articular chondrocytes (HACs) treated with IL-1β to examine its impact on cellular viability, apoptosis, inflammatory responses, and autophagy. Functional assays including Cell Counting Kit-8, flow cytometry, enzyme-linked immunosorbent assay, and commercial kits were employed to assess biochemical changes. Interaction between YTH N6-methyladenosine RNA binding protein 2 (YTHDF2) and SETD7 was explored using RNA immunoprecipitation and co-immunoprecipitation assays. SETD7 was overexpressed in OA cartilage compared with controls and increased further upon IL-1β treatment. Knockdown of SETD7 in IL-1β-treated HACs improved cellular viability, decreased apoptosis, and reversed the adverse effects on lactate dehydrogenase release and inflammatory markers (tumor necrosis factor-alpha and interleukin-6) while enhancing antioxidant enzymes (catalase, malondialdehyde, and superoxide dismutase). Additionally, autophagy was restored, as evidenced by changes in the levels of autophagy related 5, Beclin1, and sequestosome 1. Interfering with autophagy using chloroquine negated the protective effects of SETD7 knockdown. Furthermore, YTHDF2 was found to stabilize SETD7 mRNA, influencing its expression and enhancing IL-1β-induced chondrocyte injury. SETD7 plays a critical role in the pathogenesis of OA by modulating chondrocyte survival, apoptosis, inflammation, and autophagy. The interaction between YTHDF2 and SETD7 exacerbates chondrocyte injury under inflammatory conditions, highlighting potential therapeutic targets for OA treatment. The YTHDF2/SETD7 axis offers a novel insight into the molecular mechanisms governing cartilage degeneration in OA.

LINC02987 suppression hepatocellular carcinoma progression by modulating autophagy via the miR-338-3p/ATG12 axis.

Hepatocellular carcinoma (HCC), the most common primary liver cancer, is marked by a high mortality rate, with the misregulation of long non-coding RNAs (LncRNAs) playing a key role in its development. Here, we studied the role of LINC02987 in HCC. We employed bioinformatics tools to identify LncRNAs and miRNAs that exhibit differential expression in HCC. Quantitative real-time reverse transcription PCR (RT-qPCR) and Western blot analysis were utilized to quantify gene and protein expression levels. The interaction between miR-338-3p and LINC02987 or ATG12 was confirmed through dual-luciferase reporter and RNA immunoprecipitation (RIP) assays. We observed that LINC02987 was overexpressed in HCC tumor tissues and cell lines. Silencing of LINC02987 led to a reduction in cell viability, diminished clonogenic potential, and attenuated invasive and migratory capabilities. Also, decreasing protein level and fluorescence intensity of the autophagy-associated LC3 I/II. In HCC, miR-338-3p expression was downregulated, while inversely correlates with the overexpression of the autophagy protein ATG12. Mimicking miR-338-3p suppresses the activity of both LINC02987 and ATG12, as evidenced by reduced luciferase signals in corresponding reporter assays. Mimicking miR-338-3p suppresses the activity of both LINC02987 and ATG12, as evidenced by reduced luciferase signals in reporter assays. Transfection with si-LINC02987 decreased ATG12 expression, an effect that was partially reversed by miR-338-3p knockdown. Inhibition of miR-338-3p or overexpression of ATG12 increased LC3 I/II protein levels. Our results indicate that LINC02987 sequesters miR-338-3p, leading to increased ATG12 and promoting autophagy in HCC cells. These results highlight the potential of LINC02987 as a therapeutic target for the treatment of HCC.

Integrated analysis of miRNA and mRNA expression profiles in the bursa of Fabricius of specific pathogen-free chickens infected with avian reticuloendotheliosis virus strain SNV.

Reticuloendotheliosis virus (REV) is a gamma retrovirus that can cause immunosuppression, dwarf syndrome and acute reticulocytoma in poultry. The molecular mechanism by which REV infection leads to immunosuppression and tumorigenesis is poorly understood. In this study, we elucidated the regulatory network of miRNA-mRNA and the major signaling pathways involved in REV-SNV infection. Therefore, we used the spleen necrosis virus (SNV) model of REV to inoculate one-day-old specific pathogen-free (SPF) chickens and then performed global miRNA and mRNA expression profiling by conducting high-throughput sequencing of 18 bursa of Fabricius samples collected at 7, 14, and 21 dpi. In total, 213 differentially expressed miRNAs (DEMs) and 3311 differentially expressed genes (DEGs) were identified. In the miRNA-mRNA network constructed based on the association analysis of these DEMs and DEGs, 1376 negatively correlated miRNA-mRNA pairs were identified; among them, 82 pairs were identified at 7 dpi, 203 pairs were identified at 14 dpi, and 873 pairs were identified at 21 dpi. Moreover, the results of the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of the mRNAs in the network revealed greater enrichment of immune-related pathways, such as the immune system, signal transduction, cell growth and death, and signaling molecules and interactions. We confirmed the selected immune-related DEMs and their DEGs by conducting quantitative RT-PCR (qRT-PCR) analysis. These findings increased our understanding of the interactions of miRNAs and their target genes during infection with REV-SNV, and contributed to the understanding of host-virus interactions.

Aged garlic extract enhances the production of β‑defensin 4 via activation of the Wnt/β‑catenin pathway in mouse gingiva.

Periodontal disease is recognized as a chronic multifactorial inflammatory condition initiated by dysbiosis within subgingival plaque biofilms. Antimicrobial peptides exhibit a wide spectrum of antimicrobial action, and thus, provide one of the first lines of host defense against oral pathogens. Aged garlic extract (AGE) is effective for preventing the progression of periodontal disease. The present study examined whether AGE affects the production of antimicrobial peptides in mouse gingiva. Reverse transcription-quantitative PCR analysis demonstrated that oral administration of AGE in mice increased the mRNA level of Defb4 in gingival tissue, while the levels of Defb1, Defb14 and Cramp remained unaffected. AGE also upregulated the protein levels of β-defensin 4. To explore the underlying mechanism of the increased β-defensin 4 production induced by AGE, a comprehensive phosphoproteomic analysis in gingival tissues was performed. Proteomic profiling revealed activation of the canonical Wnt/β-catenin pathway in gingiva of mice treated with AGE. Treatment of mouse gingival epithelial GE1 cells with AGE resulted in an increase of β-defensin 4 in the culture medium. In support of proteomics experiments, LF3, a specific inhibitor of Wnt/β-catenin signaling, suppressed the AGE-induced production of β-defensin 4. In addition, β-catenin protein was found to accumulate within the nucleus in cells treated with AGE. In conclusion, the present findings suggested that AGE enhanced the production of β-defensin 4 in mouse gingiva through the canonical Wnt signal transduction pathway.

CCAAT/Enhancer-Binding Protein β (C/EBPβ) Regulates Calcium Deposition in Smooth Muscle Cells.

Calcium deposition in vascular smooth muscle cells (VSMCs), a form of ectopic ossification in blood vessels, can result in rigidity of the vasculature and an increase in cardiac events. Here, we report that CCAAT/enhancer-binding protein beta (C/EBPβ) potentiates calcium deposition in VSMCs and mouse aorta induced by inorganic phosphate (Pi) or vitamin D3. Based on cDNA microarray and RNA sequencing data of Pi-treated rat VSMCs, C/EBPβ was found to be upregulated and thus selected for further evaluation. Quantitative RT-PCR and Western blot analysis confirmed that C/EBPβ was upregulated in Pi-treated A10 cells, a rat VSMC line, as well as vitamin D3-treated mouse aorta. The overexpression of C/EBPβ in A10 cells increased bone runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP), and osteopontin (OPN) mRNA in the presence of Pi, as well as potentiating the Pi-induced increase in calcium contents. The Runx2 expression was increased by C/EBPβ through Runx2 P2 promotor. Our results suggest that a Pi-induced increase in C/EBPβ is a critical step in vascular calcification.

Therapeutic Targets and Natural Product Screening for Cognitive Impairments Associated with Ferroptosis in Wilson's Disease.

Wilson's disease (WD) is a hereditary condition marked by abnormalities in copper metabolism, which precipitate a spectrum of neurological symptoms and cognitive impairments. Emerging research has highlighted ferroptosis (FPT) as a distinct type of programmed cell death, potentially linked to various cognitive dysfunctions. Nevertheless, the connection between FPT and cognitive impairment in Wilson's disease (WDCI) remains largely enigmatic. In our study, we utilized a multifaceted approach, combining reverse network pharmacology, data mining, and molecular docking techniques to explore the potential for treating WDCI via FPT-related pathways. This thorough analysis revealed a series of proteins, including P38[Formula: see text], GSK3[Formula: see text], P53, GPX4, and PTGS2, as pivotal targets for WDCI treatment. Notably, Diosgenin (DG) has been identified as a prospective core component in this therapeutic framework. In the WD copper-loaded rat model, evaluations using the Morris water maze (MWM), Y maze, hematoxylin and eosin staining, transmission electron microscopy (TEM), and immunofluorescence (IF) detection showed that DG significantly enhanced cognitive function recovery, reduced structural damage to hippocampal neurons, and protected mitochondrial integrity. In addition, Western blot (WB) and quantitative reverse transcription PCR (qRT-PCR) analysis showed that DG significantly upregulated the expression levels of proteins and mRNA such as P38[Formula: see text], GSK3[Formula: see text], P53, GPX4, and PTGS2 in animal and cell models. Furthermore, DG effectively reversed the dysregulated expression of oxidative stress markers, including [Formula: see text], malondialdehyde (MDA), superoxide dismutase (SOD), and reactive oxygen species (ROS). This study elucidates the neuroprotective effect of DG on hippocampal neurons by activating the P38[Formula: see text]-mediated FPT pathway, highlighting its efficacy as a potent monomer in traditional Chinese medicine and illuminating its potential role in the clinical treatment of WDCI.

N6-methyladenosine (m6A)-circHECA from secondary hair follicle of cashmere goats: identification, regulatory network and expression regulated potentially by methylation of its host gene promoter.

The objective of this study was to identify the N6-methyladenosine (m6A)- circHECA molecule in secondary hair follicles (SHFs) of cashmere goats, and generate its potential regulatory network, as well as explore the potential relationship between transcriptional pattern of m6A-circHECA and promoter methylation of its host gene (HECA). The validation of circHECA m6A sites was performed using methylation immunoprecipitation (Me-RIP) along with reverse transcription-quantitative polymerase chain reaction (RT-qPCR) technique. The nucleus and cytoplasm localizations of m6AcircHECA were performed using SHF stem cells of cashmere goats with RT-qPCR analysis. Based on in-silico analysis, the regulatory networks of m6A-circHECA were generated with related signal pathway enrichment. The methylation level of promoter region of m6A-circHECA host gene (HECA) was assessed by the bisulfite sequencing PCR (BSPPCR) technique. The m6A-circHECA was confirmed to contain four m6A modification sites including m6A-213, m6A-297, m6A-780, and m6A-927, and it was detected mainly in cytoplasm of the SHF stem cells of cashmere goats. The integrated regulatory network analysis showed directly or indirectly complex regulatory relationships between m6A-circHECA of cashmere goats and its potential target molecules: miRNAs, mRNAs, and proteins. The regulatory network and pathway enrichment indicated that m6A-circHECA might play multiple roles in the SHF physiology process of cashmere goats through directly or indirectly interacting or regulating its potential target molecules. A higher methylation level of promoter region of HECA gene in SHFs of cashmere goats might cause the lower expression of m6A-circHECA. The m6A-circHECA might play multiple roles in SHF physiology process of cashmere goats through miRNA mediated pathways along with directly or indirectly interaction with its target proteins. The promoter methylation of m6A-circHECA host gene (HECA) most likely was implicated in its expression inhibition in SHFs of cashmere goats.

Fluorescent Staining-Assisted Evaluation of the Anticancer Potential of Nyctanthes arbor-tristis Extracts in HepG2 Liver Cancer Cells.

The current study's initial goal was to investigate the anticancer activity of the extracts from the flowering plant Nyctanthes arbor-tristis. The ethanol extracts' phytochemical analysis was qualitatively determined. It was determined that the various phytochemical substances had antibacterial, antioxidant, and anticancer effects. GC-MS analysis revealed compounds like maleic anhydride, butanedioic acid, and oxalic acid emphasize the antioxidant and anticancer properties. MTT assay was investigated against three different cell lines (HepG2, A549, and MCF7). High level of resistance to HepG2 liver cancer cell lines was noted with ethanol flower extracts with IC50 value of 67.98 μg/mL. EtBr staining assay revealed HepG2 apoptotic cells displaying nuclear and membrane alterations when exposed to 70 μg/mL of ethanol extract. The cells treated with 70 μg/mL of extract showed a reduction of mitochondrial membrane potential, as evidenced by the MMP assay, which showed red fluorescence decreasing and green fluorescence increasing. DNA fragmentation analysis showed HepG2 cancer cells showed no signs of DNA fragmentation after being treated with 50 μM treatments of flower extracts for 72 h. Quantitative RT-PCR analysis showed increased Bcl2 gene expression with fold variation level of about 0.086094579. The obtained results emphasized the significance of Nyctanthes arbor-tristis extracts with a suggestion of chemotherapeutic medication to treat various cancers in the future.