Cell Scratch Assay Research Articles

IRF7-deficient MDBK cell based on CRISPR/Cas9 technology for enhancing IBRV replication

Infectious bovine rhinotracheitis (IBR), characterized by acute respiratory lesions in cattle, is a major infectious disease caused by bovine alphaherpesvirus-1 (BoAHV-1). Control of this disease is primarily depending on vaccination. Madin-Darby bovine kidney cells (MDBK) being the main host cells and the important production platform for IBR vaccines. However, innate immune genes inhibit viral replication. Accordingly, the aim of this study was developing of IRF7 gene deleted MDBK cells to facilitate the production of high-titer vaccines. The CRISPR/Cas9 technology was used to knock out the IRF7 gene in MDBK cells and the impact on virus replication was examined using virus growth curves, CCK-8 assays, cell scratch assays, and qPCR. The knockout of the IRF7 gene in MDBK cells led to an increased replication capacity of IBRV and a significant reduction in type I interferons expression, specifically IFN-α and IFN-β. This indicates that IRF7−/−MDBK cell lines can effectively result in production of IBRV with high-titer, which will enhance the development of inactivated or attenuated vaccines.

Triptolide induces apoptosis of glioma cells by inhibiting NF-κB activation during oxidative stress

Glioma is a common and fatal malignant primary brain tumor. Radiotherapy and first-line chemotherapy have little effect on the survival rate of patients, requiring alternative therapies. The main active ingredient of Tripterygium wilfordii Hook. F. triptolide (TP) has been shown to have anti-inflammatory and anti-proliferative properties, along with a wide range of anticancer activities. This study aimed to investigate the molecular mechanisms of triptolide in glioma treatment through network pharmacology and experimental validation. Cell viability was first assessed using Cell Counting Kit-8 (CCK8), followed by cell scratch assay and cell migration ability. Apoptosis-related markers, including TUNEL staining, Bcl-2-associated X protein (Bax), and B-cell lymphoma-2 (Bcl-2), were detected. Network pharmacology was used to predict the key targets of glioma, detect its signal pathways, screen the key components and targets for molecular docking, and explore the signaling pathways of TP. Lastly, immunofluorescence assays and ELISA were performed to elucidate the underlying mechanistic pathways. The network pharmacology data suggested that TP may inhibit glioma proliferation by regulating the signaling pathway of the nuclear factor kappa-B (NF-κB). The results showed that the underlying mechanism involved the regulation of the NF-κB signaling pathway to promote the generation of reactive oxygen species, thereby enhancing oxidative stress response and promoting cell apoptosis.

Long non-coding RNA HOTAIR promotes tumorigenesis by affecting proliferation, invasion, migration and apoptosis of liver cancer cells.

Increasing evidence shows that Hox transcript antisense RNA (HOTAIR) plays a vital role in liver cancer initiation and progression by affecting the proliferation, invasion, migration and apoptosis of liver cancer cells. However, the underlying mechanism of how HOTAIR exerts its functions in liver cancer cells remains unclear. Previous studies have shown that HOTAIR affects the invasion and migration of liver cancer cells by regulating the expression of E-cadherin. Snail2, a transcription factor involved in epithelial-mesenchymal transition, directly binds to the E-boxes of theE-cadherinpromoter to repress its transcription. The aim of the study was to examine the correlation between HOTAIR and Snail2 in the HOTAIR/Snail2/E-cadherin signal pathway and explore the role of HOTAIR in the proliferation, invasion, migration and apoptosis of liver cancer cells. 50 matched normal liver tissues and 373 liver cancer tissues were analysed and evaluated. HepG2 and SNU-387 cells were cultured and transfected with plasmids knocking down HOTAIR to disrupt HOTAIR expression. Cell scratch and transwell assays were performed to examine the migration and invasion of HepG2 and SNU-387 cells; in addition, the expression of MMP2 and MMP9 was detected by immunoblotting analysis, RT-qPCR analysis, immunofluorescence analysis, and bioinformatics analysis, which elucidated the regulatory relationship between HOTAIR and Snail2. We used flow cytometry and JC-1 probe analysis assays to clarify the function of HOTAIR in liver cancer cell apoptosis. The HOTAIR mRNA was upregulated in liver cancer tissues, which was related to worse overall survival. HOTAIR induced the expression of matrix metalloproteinase-9 (MMP9) and metalloproteinase-2 (MMP2), leading to degradation of extracellular matrix. HOTAIR knockdown significantly reduced the doubling time and inhibited cell migration and invasion of liver cancer cells. Furthermore, HOTAIR depletion induced mitochondrial-related apoptosis in HepG2 and SNU-387 cell lines. In this study, we proposed a novel mechanism in which HOTAIR promotes invasion and migration of liver cancer cells by regulating the nuclear localization of Snail2.

Nerve Growth Factor from Pancreatic Cancer Cells Promotes the Cancer Progression by Inducing Nerve Cell-Secreted Interleukin-6.

Pancreatic cancer (PC) is a cancer with a poor prognosis, and nerve growth factor (NGF) is involved in the pathogenesis of PC within the unknown exact role. Herein, SW1990 cells and PC12 cells were co-cultured using transwell co-culture system and subsequently revealed that NGF was overexpressed in SW1990 cells and promoted PC12 cell proliferation. Knockdown of NGF expression in SW1990 cells using lentiviral shRNA effectively inhibited NGF expression in SW1990 cells and reduced its stimulatory effect on PC12 cell proliferation. Additionally, NGF in SW1990 cells increased the expression of IL-6, dopamine, and c-FOS, as well as decreased the level of lactate dehydrogenase, in PC12 cells, whereas the inhibition of NGF expression significantly reduced the levels of IL-6, dopamine and c-FOS, indicating the critical role of IL-6/STAT3 signaling in PC progression. Finally, cell proliferation, migration, and invasion were assessed using cell counting kit-8, scratch, and Transwell assays, which showed that activated neurons promoted the proliferation, migration, invasion, and NGF secretion of SW1990 cells through the IL-6/STAT3 pathway. The results revealed that NGF secreted by PC cells played a pivotal role in PC progression via regulating activated neural cells-secreted IL-6, providing new theoretical insights for the treatment of PC.

Clinical Significance of HHLA2 as a Novel Therapeutic Target for Colorectal Cancer.

Colorectal cancer (CRC) is a high-indence malignance of the digestive system with a high mortality rate in the world. The results are desired to provide an important theoretical basis for discovering new therapeutic targets for CRC. The expression of human endogenous retrovirus-H-long terminal repeat association protein 2 (HHLA2) in human CRC was detected to explore its correlationship with clinicopathological features and prognosis of patients and its potential in treating CRC. Western blot was employed to detect HHLA2 expression in fresh tissues obtained from 6 CRC patients' excisions, including cancer, paracancer, and normal issues. Immunohistochemical staining was employed to determine HHLA2 expression in paraffin-embedded specimens of 139 patients with colorectal cancer, and its relationship with the clinicopathological profiles and survival was analyzed. Small interfering RNA (siRNA) targeting HHLA2 was used to transfect CRC cells to silent HHLA2. MTT, plate colony formation, cell scratch, and Transwell assay were conducted to observe the proliferation, migration, and invasion of CRC cells. HHLA2 protein was expressed in human colorectal cancer tissues, paracancer tissues and normal tissues, which was significantly upregulated in cancer tissues (P<0.01). HHLA2 expression level in CRC tissues showed a close correlationship with the invasion depth of the tumor (P=0.000), metastasis of regional lymph nodes (P=0.018), clinical stage (P=0.010), and patient survival (P=0.011). Correlation with gender (P=0.873), age (P=0.864), location of the tumor (P=0.768), degree of tumor differentiation (P=0.569) and distant metastasis (P=0.494) exhibited no significance. The survival time of CRC patients with high and low HHLA2 expression groups was 43.231 months and 55.649 months, respectively, with a statistical difference between the two groups (P=0.001). Silencing HHLA2 inhibited proliferation, migration and invasion of CRC cells significantly. HHLA2 is overexpressed in CRC tissues which is associated with poor prognosis of patients. HHLA2 might be recognized as a new candidate for adjuvant diagnosis and prognosis of CRC, as well as a promised new target for immunotherapy of CRC.

Machine learning and single-cell RNA sequencing reveal relationship between intratumor CD8+ T cells and uveal melanoma metastasis

PurposeUveal melanoma (UM) is adults’ most common primary intraocular malignant tumor. It has been observed that 40% of patients experience distant metastasis during subsequent treatment. While there exist multigene models developed using machine learning methods to assess metastasis and prognosis, the immune microenvironment’s specific mechanisms influencing the tumor microenvironment have not been clarified. Single-cell transcriptome sequencing can accurately identify different types of cells in a tissue for precise analysis. This study aims to develop a model with fewer genes to evaluate metastasis risk in UM patients and provide a theoretical basis for UM immunotherapy.MethodsRNA-seq data and clinical information from 79 μm patients from TCGA were used to construct prognostic models. Mechanisms were probed using two single-cell datasets derived from the GEO database. After screening for metastasis-related genes, enrichment analysis was performed using GO and KEGG. Prognostic genes were screened using log-rank test and one-way Cox regression, and prognostic models were established using LASSO regression analysis and multifactor Cox regression analysis. The TCGA-UVM dataset was used as internal validation and dataset GSE22138 as external validation data. A time-dependent subject work characteristic curve (time-ROC) was established to assess the predictive ability of the model. Subsequently, dimensionality reduction, clustering, pseudo-temporal analysis and cellular communication analysis were performed on GSE138665 and GSE139829 to explore the underlying mechanisms involved. Cellular experiments were also used to validate the relevant findings.ResultsBased on clinical characteristics and RNA-seq transcriptomic data from 79 samples in the TCGA-UVM cohort, 247 metastasis-related genes were identified. Survival models for three genes (SLC25A38, EDNRB, and LURAP1) were then constructed using lasso regression and multifactorial cox regression. Kaplan-Meier survival analysis showed that the high-risk group was associated with poorer overall survival (OS) and metastasis-free survival (MFS) in UM patients. Time-dependent ROC curves demonstrated high predictive performance in 6 m, 18 m, and 30 m prognostic models. Cell scratch assay showed that the 24 h and 48 h migration rates of cells with reduced expression of the three genes were significantly higher than those of the si-NC group. CD8 + T cells may play an important role in tumour metastasis as revealed by immune infiltration analysis. An increase in the percentage of cytotoxic CD8 + T cells in the metastatic high-risk group was found in the exploration of single-cell transcriptome data. The communication intensity of cytotoxic CD8 was significantly enhanced. It was also found that the CD8 + T cells in the two groups were in different states, although the number of CD8 + T cells in the high-risk group increased, they were mostly in the exhausted and undifferentiated state, while in the low-risk group, the CD8 + T cells were mostly in the functional state.ConclusionsWe developed a precise and stable 3-gene model to predict the metastatic risk and prognosis of patients. CD8 + T cells exhaustion in the tumor microenvironment play a crucial role in UM metastasis.

CXC chemokine receptor 4 - mediated immune modulation and tumor microenvironment heterogeneity in gastric cancer: Utilizing multi-omics approaches to identify potential therapeutic targets.

G-protein-coupled receptors (GPRs) are critical regulators of various biological behaviors, and their role in gastric cancer (GC) progression is gaining increasing attention. Among them, the immune regulatory mechanisms mediated by chemokine receptor 4 (CXCR4) remain insufficiently understood. This study aims to explore the immune regulatory functions of CXCR4 and the heterogeneity of the tumor microenvironment (TME) by examining GPR-related gene expression in GC. Through multi-omics approaches, including spatial transcriptomics and single-cell RNA sequencing, we investigated the oncogenic mechanisms of CXCR4, particularly its role in T cell immune exhaustion. In vitro experiments, including ELISA, PCR, CCK8 assays, cell scratch assays, and colony formation assays, were used to validate the role of CXCR4 in the migration and invasion of AGS and SNU-1 cell lines. CXCR4 silencing using siRNA further demonstrated its regulatory effects on these cellular processes. Our results revealed a strong correlation between elevated CXCR4 expression and increased exhaustion of regulatory T cells (Tregs) in the TME. Furthermore, heightened CXCR4 expression was linked to increased TME heterogeneity, driven by oxidative stress and activation of the NF-κB pathway, promoting immune evasion and tumor progression. Silencing CXCR4 significantly inhibited the invasive and proliferative abilities of AGS and SNU-1 cells, while also reducing the expression of pro-inflammatory cytokines IL-1β and interleukin-6, thus alleviating chronic inflammation and improving TME conditions. In conclusion, our comprehensive investigation highlights CXCR4 as a key mediator of TME dynamics and immune modulation in GC. Targeting CXCR4 presents a promising therapeutic strategy to slow tumor progression by reducing Tregs-mediated immune exhaustion and TME heterogeneity, positioning it as a novel therapeutic target in GC treatment.

Cisplatin promotes TNF-α autocrine to trigger RIP1/RIP3/MLKL-dependent necroptosis of human head and neck squamous cell carcinoma cells

To investigate whether cisplatin induces tumor necrosis factor-α (TNF-α) secretion in human head and neck squamous cell carcinoma (HNSCC) cells to trigger RIP1/RIP3/MLKL-dependent necroptosis of the cells. HNSCC cell lines HN4 and SCC4 treated with cisplatin (CDDP) or the combined treatment with CDDP and z-VAD-fmk (a caspase inhibitor) or Nec-1 (a necroptosis inhibitor) for 24 h were examined for changes in cell viability using CCK8 assay and expressions of caspase-8 and necroptosis pathway proteins (RIP1/RIP3/MLKL) using Western blotting. The changes in migration of the cells were assessed with cell scratch assay, and the expressions of epithelial-mesenchymal transition (EMT) marker proteins N-cadherin, vimentin, and E-cadherin as well as the expressions of NF-κB (p65) and TNF-α were detected with Western blotting. The IC50 of cisplatin was 10 μg/mL in HN4 cells and 15 μg/mL in SCC4 cells. Cisplatin treatment significantly decreased the expressions of caspase-8, N-cadherin and vimentin and increased the expressions of Ecadherin, the necroptosis pathway proteins (RIP1/RIP3/MLKL), TNF-α, and NF-κB (p65), and these changes were obviously inhibited by treatment with Nec-1. Cisplatin stimulation also significantly lowered migration of the cells, and this inhibitory effect was strongly attenuated by Nec-1 treatment. Cisplatin activates nuclear factor-κB signaling in HNSCCs to promote TNF-α autocrine and induce RIP1/RIP3/MLKL-dependent necroptosis, thus leading to inhibition of cell proliferation.

Melanoma-inhibiting activity promotes the migration and odontoblastic differentiation of stem cells of apical papilla

ObjectiveMelanoma Inhibitory Activity (MIA) has been predominantly studied in the context of melanoma and cartilage development. However, its role in dental pulp development and stem cell behavior remains largely unexplored. This study investigates the expression pattern of MIA in dental pulp tissues and its potential role in the proliferation, migration, and odontoblastic differentiation of stem cells from the apical papilla (SCAPs). DesignMIA expression in human pulp tissue was demonstrated by immunohistochemistry. SCAPs were cultured in normal and mineralization induction media, with MIA levels monitored via RT-qPCR and Western blot. Cell proliferation was evaluated using the CCK8 assay, while transwell and cell scratch assays were conducted to examine cell migration. The effect of MIA on odontoblastic differentiation was examined by qRT-PCR, Alkaline phosphatase activity assay, and Western blot. siRNA was used to knock down MIA to investigate its effect. A mouse subcutaneous implantation model was used to assess whether MIA promotes odontoblastic differentiation in vivo. ResultsMIA expression was observed in the papilla and odontoblasts layer of the developing pulp. In vitro, MIA expression increased during SCAPs differentiation and was found to significantly enhance migration, and odontoblastic differentiation but not proliferation. Gene knockdown experiments confirmed MIA’s pivotal role in promoting SCAPs migration and differentiation. In vivo, MIA facilitated the formation of dentin-like structures and enhanced pulp-dentin complex regeneration. ConclusionMIA plays a crucial role in SCAPs’ migration and differentiation, suggesting its potential application in pulp-dentin regeneration therapies. Further studies are required to fully elucidate the underlying mechanisms.

The long non-coding RNA NEAT1 contributes to aberrant STAT3 signaling in pancreatic cancer and is regulated by a metalloprotease-disintegrin ADAM8/miR-181a-5p axis.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers and several studies demonstrate that STAT3 has critical roles throughout the course of PDAC pathogenesis. TCGA, microarray, and immunohistochemistry data from a PDAC cohort were used for clinical analyses. Panc89 cells with ADAM8 knockout, re-expression of ADAM8 mutants, and Panc1 cells overexpressing ADAM8 were generated. Gene expression analyses of ADAM8, STAT3, long non-coding (lnc) RNA NEAT1, miR-181a-5p and ICAM1 were performed by quantitative PCR. Subcellular fractionation quantified NEAT1 expression in cytoplasm and nucleus of PDAC cell lines. Cell proliferation, scratch, and invasion assays were performed to detect growth rate, migration and invasion capabilities of cells. Gain and loss of function experiments were carried out to investigate the biological effects of lncRNA NEAT1 and miR-181a-5p on PDAC cells and downstream genes. Dual-luciferase reporter gene assay determined interaction and binding sites of miR-181a-5p in lncRNA NEAT1. Pull down assays, RNA binding protein immunoprecipitation (RIP), and ubiquitination assays explored the molecular interaction between lncRNA NEAT1 and STAT3. High ADAM8 expression causes aberrant STAT3 signaling in PDAC cells and is positively correlated with NEAT1 expression. NEAT1 binding to STAT3 was confirmed and prevents STAT3 degradation in the proteasome as increased degradation of STAT3 was observed in ADAM8 knockout cells and cells treated with bortezomib. Furthermore, miRNA-181a-5p regulates NEAT1 expression by direct binding to the NEAT1 promoter. ADAM8 regulates intracellular STAT3 levels via miR-181a-5p and NEAT1 in pancreatic cancer.

Fabrication and Properties of Hydrogel Dressings Based on Genipin Crosslinked Chondroitin Sulfate and Chitosan.

Multifunctional hydrogel dressings remain highly sought after for the promotion of skin wound regeneration. In the present study, multifunctional CHS-DA/HACC (CH) hydrogels with an interpenetrated network were constructed using hydroxypropyl trimethyl ammonium chloride modified chitosan (HACC) and dopamine-modified chondroitin sulfate (CHS-DA), using genipin as crosslinker. The synthesis of HACC and CHS-DA was effectively confirmed using Fourier transform infrared (FT-IR) analysis and 1H nuclear magnetic resonance (1H NMR) spectroscopy. The prepared CH hydrogels exhibited a network of interconnected pores within the microstructure. Furthermore, rheological testing demonstrated that CH hydrogels exhibited strong mechanical properties, stability, and injectability. Further characterization investigations showed that the CH hydrogels showed favorable self-healing and self-adhesion properties. It was also shown that increasing HACC concentration ratio was positively correlated with the antibacterial activity of CH hydrogels, as evidenced by their resistance to Escherichia coli and Staphylococcus aureus. Additionally, Cell Counting Kit-8 (CCK-8) tests, fluorescent images, and a cell scratch assay demonstrated that CH hydrogels had good biocompatibility and cell migration ability. The multifunctional interpenetrated network hydrogels were shown to have good antibacterial properties, antioxidant properties, stable storage modulus and loss modulus, injectable properties, self-healing properties, and biocompatibility, highlighting their potential as wound dressings in wound healing applications.

Extracellular vesicles from inflammatory-stimulated BMSCs ameliorate osteoarthritis via Rpl14 mediated synovial macrophage polarization

Bone marrow mesenchymal stem cells (BMSCs) and extracellular vesicles (EVs) were considered promising methods for the treatment of diverse diseases, including osteoarthritis (OA). However, the role of inflammatory-stimulated BMSCs driven EVs (iBMSC-EVs) in OA remains unclear. This study aims to investigate the potential function of iBMSC-EVs in ameliorating OA and its underlying molecular mechanism. In this study, EVs were isolated from inflammatory-stimulated BMSCs and destabilization of the medial meniscus (DMM) surgery was performed to induce knee OA model in mice. Micro-CT, H&E Staining, Safranin O-Fast Green Staining and immunofluorescence were used to examine the in vivo therapeutic effect of iBMSC-EVs on OA. Public single-cell RNA-seq data was downloaded and bulk RNA-seq data was acquired to identify critical genes promoted by iBMSC-EVs in macrophage polarization during OA. Real-time quantitative polymerase chain reaction (RT-qPCR), western blot, enzyme-linked immunosorbent assay (ELISA), cell counting kit-8 (CCK-8), transwell and scratch assays were used to further confirm the in vitro effect of iBMSC-EVs on macrophage polarization and chondrocyte proliferation, migration, as well as ECM anabolism and catabolism. In vivo experiments demonstrated that iBMSC-EVs could alleviate OA in mice and promote the reprogramming of M1 macrophages towards an M2 phenotype. RNA-Seq and scRNA-Seq analyses indicated that iBMSC-EVs altered the expression profiles of OA-related genes, with Rpl14 identified as a critical gene regulating macrophage reprogramming in OA. In vivo and in vitro experiments further confirmed that iBMSC-EVs, by downregulating Rpl14 expression, promoted M1 macrophage reprogramming to M2, facilitating chondrocyte proliferation and migration, inhibiting ECM degradation and ultimately alleviating OA progression. In conclusion, our data revealed the substantive role of iBMSC-EVs in ameliorating OA progression via Rpl14 mediated synovial macrophage polarization, presenting a promising therapeutic method for the treatment of OA.

MicroRNA-503 Suppresses Oral Mucosal Fibroblast Differentiation by Regulating RAS/RAF/MEK/ERK Signaling Pathway.

The abnormal proliferation and differentiation of oral mucosal fibroblasts (FBs) is the key to the progression of oral submucosal fibrosis. To clarify the mechanism of platelet-derived growth factor (PDGF-BB)-induced FBs fibrosis in oral mucosa, real-time quantitative polymerase chain reaction and Western blot were used in this study to detect the expression of miR-503 and the expression of p-MEK, p-ERK, miR-503, RAF, smooth actin and type I collagen under different time and concentration stimulation of PDGF-BB. The effects of overexpression of miR-503 or RAF on the proliferation and migration of FBs were detected by cell counting kit 8 and cell scratch assay, respectively. A dual luciferase reporter gene assay was used to verify the targeting effect of miR-503 on RAF. The results showed that miR-503 was downregulated in a dose- and time-dependent manner in PDGF-BB-induced FBs. In addition, RAF is a direct target of miR-503 and can be negatively regulated. Overexpression of RAF can promote FB proliferation, migration, differentiation, collagen synthesis, and activation of downstream molecules (MEK/ERK), while overexpression of miR-503 can partially reverse the effects of RAF. Therefore, miR-503 regulates the biological behavior of PDGF-BB-induced oral mucosal FBs by influencing the activation of the RAS/RAF/MEK/ERK signaling pathway.

Polyhydroxybutyrate/poly(ε‐caprolactone)‐based electrospun membranes loaded with amoxicillin‐potassium clavulanate halloysite nanotubes for biomedical applications

AbstractBiopolymers exhibit distinct properties for biomedical applications. Different biopolymer classes are utilized for various applications, for example antibacterial properties, drug delivery, tissue engineering, tissue scaffolds etc. In the present investigation, a nano‐bioengineering approach was followed to prepare polyhydroxybutyrate and polycaprolactone polymer‐based drug‐loaded halloysite nanotube electrospun membranes for biomedical applications. Functionalized halloysite nanotubes ((3‐aminopropyl)triethoxysilane acid treated halloysite nanotubes) at different weight percentages (1, 3, 5 and 7 wt%) were loaded with a broad‐spectrum antibiotic amoxicillin trihydrate‐potassium clavulanate, incorporated into the electrospun membranes, and characterized by different techniques such as XRD, FTIR, SEM, TEM and TGA. Different physical and mechanical properties were evaluated, such as porosity, water uptake, water vapor transmission rate, wettability and tensile properties. The developed membranes exhibited good in vitro biological properties, for example antibacterial, effective cell migration and less toxicity, as confirmed by disk diffusion, MTT (3‐(4,5‐dimethylthiazolyl‐2)‐2,5‐diphenyltetrazolium bromide) and cell scratch assays. A sustained drug release profile was observed from all the developed membranes. Overall results on the characterization of the developed membranes confirm the suitability of their use for different biomedical applications and as a wound dressing application. © 2024 Society of Chemical Industry.

HLTF/SERPINE1 Axis Plays a Crucial Pro-Oncogenic Role in the Progression from Cervical Precancerous Lesions to Cervical Carcinoma in vitro

Objectives: Cervical carcinoma (CC) is prevalent among women worldwide with increasing risk. Finding effective methods for treating CC is of utmost importance. The aim of this study was to investigate the effect of SERPINE1 on the progression of cervical precancerous lesions to CC. Design: This study used transcriptome sequencing and in vitro cell line. Participants/Materials: Cervical precancerous lesions and CC samples and human cervical epithelial immortalized cell line H8, human CC cell lines HeLa, and CaSki were involved in this study. Setting and Methods: Next-generation sequencing was applied to identify 100 differentially expressed genes from cervical precancerous lesions and CC samples. With the application of the Search Tool for the Retrieval of Interacting Genes (STRING) database, we carried out the protein-protein interaction network analysis, thus screening out serine protease inhibitor clade E member 1 (SERPINE1) with significant upregulation in CC cells. The helicase-like transcription factor (HLTF) was predicted as the upstream transcription factor using Human Transcription Factor Database (HumanTFDB). The chromatin immunoprecipitation (ChIP) experiment was conducted to validate the interaction between SERPINE1 and HLTF. The immunohistochemistry was employed to determine the expression of SERPINE1 and HLTF in CC tissues. Following the upregulation or downregulation of SERPINE1 and HLTF, the real-time quantitative reverse transcription polymerase chain reaction was carried out to assess mRNA expression levels of SERPINE1 and HLTF in cells. Cell viability, migration, and invasion were evaluated using MTT assay, cell scratch assay, and Transwell assay, respectively. Western blot analysis was conducted to assess changes in the expression levels of matrix metalloproteinases and proteins related to epithelial-mesenchymal transition (EMT). Results: The ChIP experiment confirmed the interaction between HLTF and SERPINE1. HLTF and SERPINE1 were upregulated in CC tissues and cells, and silencing SERPINE1 inhibited the EMT process and viability, migration, and invasion of CC cells. However, overexpression of SERPINE1 in CC cells showed the opposite trend. Rescue experiments demonstrated that silencing HLTF repressed CC cell viability, migration, and invasion, which could be restored by overexpressing SERPINE1. Limitations: The effect of the HLTF/SERPINE1 axis on CC malignant progression has not been confirmed by in vivo experiments. Conclusion: HLTF transcriptionally activates SERPINE1, promoting the progression from cervical precancerous lesions to CC.

Phytochemical Analysis and Evaluation of Antioxidant, Antidiabetic, and Anti-inflammatory Properties of Aegle marmelos and Its Validation in an In-Vitro Cell Model.

Persistent hyperglycemia significantly increases oxidative stress and inflammation resulting in multiple cellular and molecular alterations which further exacerbate the diabetes associated complications.Aegle marmelos (L.) Corrêais a medicinal plant used in the Indian system of medicine for treating various disorders including diabetes. However, studies on phytoconstituents and their pharmacological activity of this plant are limited. Therefore, we aimed to determine the phytochemical components, evaluate the antidiabetic activity, anti-inflammatory activity, and antioxidant activity ofA. marmelosleaf extract, and validate its mechanistic effects in an in vitro cell model. The qualitative and quantitative analysis of the different phytoconstituents in the extract was determined using standardized protocols. The antioxidant activity of the extract was evaluated by 2,2-di-phenyl-1-picrylhydrazyl (DPPH) radical scavenging capacity assay and ferric reducing antioxidant power (FRAP) assay. The antidiabetic activity of the extract was evaluated by α-amylase inhibition and α-glucosidase inhibition assay. The anti-inflammatory activity was studied using an albumin denaturation assay. In addition, the pharmacological effect(s) of leaf extract was checked in the normal rat kidney fibroblast cells (NRK-49F) under high glucose conditions. Intracellular reactive oxygen species (ROS) generation was measured by fluorometry using fluorescence probe 2',7'-dichlorodihydrofluorescin diacetate (DCF-DA). mRNA expression of inflammatory markers including inducible nitric oxide synthase (iNOS) and tumor necrosis factor-alpha (TNF-α) was studied using real-time quantitative polymerase chain reaction (RT-qPCR). Cell migration was studied using cell scratch assay. Statistical analysis was performed using GraphPad Prism version 8.0. The phytochemical analysis ofA. marmelosleaf extract revealed the presence of alkaloids, phenols, flavonoids, and saponins. The extract showed higher antioxidant activity in the DPPH (IC50=258.21 µg/mL) and FRAP assay (IC50=293.83 µg/mL). The extract exhibited prominent antidiabetic activity by inhibiting enzymes α-Amylase (IC50=73.2 µg/mL) and α-glucosidase (IC50=43.9 µg/mL). In addition, the extract showed effective anti-inflammatory activity by significantly inhibiting the denaturation of egg albumin (IC50=102.8 µg/mL). Further, the leaf extract significantly decreased the high glucose-induced ROS generation as well as inflammatory markers in rat fibroblast cell lines in a dose-dependent manner. Additionally, high glucose-induced cell migration as the measure of cell injury was effectively reduced by the extract treatment. A. marmelosleafextract was quantified to possess a substantial amount of important phytoconstituents that have promising pharmacological properties. Besides showing antidiabetic activity, the extract significantly combats the high glucose-induced ROS generation, inflammatory markers expressions, and cell migration. Further, in-depth studies and clinical trials are warranted so as to position these traditional remedies for the treatment of metabolic disorders.

AT11-guided liposomes for oral cancer cells: From characterization towards in vitro evaluation

The increasing prevalence of oral cancers, particularly those attributed to human papillomavirus infection, along with the associated drawbacks of conventional therapies, has spurred research into more effective treatment modalities. Liposomes have emerged as a potential strategy to improve the delivery of anticancer molecules to the cells of interest. However, functionalizing these nanoparticles with targeting agents could significantly enhance their selectivity. A prospective approach involves the incorporation of aptamers, which facilitate drug accumulation within cancer cells. Among these, the AT11 aptamer has garnered attention due to its improved toxicity and high affinity to nucleolin, making it a promising targeting moiety. Therefore, we propose to use AT11 for liposomes’ functionalization, to enhance the selectivity of C8, a potential anticancer compound, specifically targeting oral cancer cells. Thus, we produced liposomes (empty or C8-associated) by ethanol injection method and, then, proceeded with their functionalization with AT11-TEG-Cholesteryl. The resulting liposomes were characterized by dynamic light scattering and hydrodynamic diameters of 130–136 nm were obtained. Additionally, the effect of the produced liposomes on the viability of squamous cell carcinoma of the tongue (UPCI-SCC-154) and nonmalignant (Het1A) cells was determined, by MTT assay. It was demonstrated that the viability of cells treated with empty liposomes was almost unaffected until the 53.6 μg/mL concentration. After treating the cells with C8-associated liposomes, both cell lines showed a dose-response effect. Moreover, the AT11-functionalization of the obtained C8-associated liposomes enhanced the selectivity of the liposomes towards oral cancer cell line, as observed by the MTT assay (43.2 % in UPCI-SCC-154 versus 79.4 % in Het1A) and confocal microscopy. Furthermore, the anticancer potential of AT11 C8-associated liposomes was evaluated in terms of proliferation (ki67 positive cells), cell death (propidium iodide internalization in non-permeabilized cells and caspase-3 activity), migration (cell scratch assay) and invasion. Overall, the produced liposomes decreased tongue cancer cell proliferation (53.6 % versus 99.4 %), migration (from 24.6 % to −3.8 %) and invasion (to 19 %) and induced cell death (11.6-fold increase in propidium iodide positive cells, and a 140 % of caspase-3 activity). These findings suggest that the AT11 C8-associated liposomes are promising drug carriers for oral cancer therapy.

LncRNA XIST/miR-455-3p/HOXC4 axis promotes breast cancer development by activating TGF-β/SMAD signaling pathway.

Breast cancer is the second primary cause of cancer death among women. Long non-coding RNA (lncRNA) X-inactive specific transcript (XIST) is a central regulator for X chromosome inactivation, and its abnormal expression is a primary feature of breast cancer. So far, the mechanism of XIST in breast cancer has not been fully elucidated. We attempted to illustrate the mechanism of XIST in breast cancer. The expressions of XIST, microRNA-455-3p (miR-455-3p) in breast cancer were measured using quantitative real-time PCR. The expressions of homeobox C4 (HOXC4) were assessed with immunohistochemical and Western blot. Also, the functions of XIST in breast cancer were assessed by Cell Counting Kit-8 analysis, colony formation assay, flow cytometry, Western blot, Transwell, and cell scratch assays. Meanwhile, the mechanism of XIST in breast cancer was validated using database analysis and dual-luciferase reporter assay. Furthermore, the function of XIST in breast cancer in vivo was estimated by tumor xenograft model, immunohistochemical assay, and hematoxylin-eosin staining. XIST and HOXC4 expressions were increased, but miR-455-3p expressions were decreased in breast cancer tissues and cells. Knocking down XIST restrained breast cancer cell proliferation, invasion, migration, epithelial-mesenchymal transformation (EMT), and induced cell cycle arrest at G0/G1. Meanwhile, XIST interacted with miR-455-3p, while miR-455-3p interacted with HOXC4. XIST knockdown repressed breast cancer cell proliferation, invasion, and EMT, while miR-455-3p inhibitor or HOXC4 overexpression abolished those impacts. HOXC4 overexpression also blocked the impacts of miR-455-3p mimic on breast cancer cell malignant behavior. In vivo experimental data further indicated that XIST knockdown repressed breast cancer cell tumorigenic ability, and decreased HOXC4 and p-SMAD3 (TGF-β/SMAD-related protein) expressions.XIST/miR-455-3p/HOXC4 facilitated breast cancer development by activating the TGF-β/SMAD pathway.

Antioxidant, Antiinflammation, and Antifibrotic Activity of Ciplukan (Physalis angulata L). Extract.

Physalis angulata Linn. (Ciplukan) is a plant widely used in traditional medicine in subtropical and tropical regions. Most studies focus on its antioxidant and anti-inflammatory activity. Many studies also reported its therapeutic potential for treating cancer, malaria, hepatitis, rheumatism, liver problems, and tumors, but few studies have reported its anti-fibrosis activity. Here, we aimed to investigate the potential of P. angulata as an antioxidant and anti-inflammatory that may be correlated with its anti-fibrosis action. In our study, we treated 3T3-L1 and TGF-β-induced 3T3-L1 cells with an ethanol extract of P. angulata. We then monitored the cell's response, evaluated the antioxidant activity using an MTT assay, and observed the cells' migration using the cell scratch assay. We used RT-PCR to determine the expression of HIF-1α and IL-6 on TGF-β-induced 3T3-L1 cells. The ethanol extract of P. angulata showed antioxidant activity and promoted cell proliferation on 3T3-L1 cells. Interestingly, the extract inhibited the migration of TGF-β-induced 3T3-L1 cells. Further analysis revealed that the extract could inhibit HIF-1α expression and suppress IL-6 expression on TGF-β-induced 3T3-L1 cells. The ethanol extract of P. angulata showed antioxidant and anti-inflammation activities in 3T3-L1 cells. Both activities are associated with the antifibrotic activity of P. angulata's ethanol extract.

Groenlandicine enhances cisplatin sensitivity in cisplatin-resistant osteosarcoma cells through the BAX/Bcl-2/Caspase-9/Caspase-3 pathway

Groenlandicine is a protoberberine alkaloid isolated from Coptidis Rhizoma, a widely used traditional Chinese medicine known for its various biological activities. This study aims to validate groenlandicine’s effect on both cisplatin-sensitive and cisplatin-resistant osteosarcoma (OS) cells, along with exploring its potential molecular mechanism.The ligand-based virtual screening (LBVS) method and molecular docking were employed to screen drugs. CCK-8 and FCM were used to measure the effect of groenlandicine on the OS cells transfected by lentivirus with over-expression or low-expression of TOP1. Cell scratch assay, CCK-8, FCM, and the EdU assay were utilized to evaluate the effect of groenlandicine on cisplatin-resistant cells. WB, immunofluorescence, and PCR were conducted to measure the levels of TOP1, Bcl-2, BAX, Caspase-9, and Caspase-3. Additionally, a subcutaneous tumor model was established in nude mice to verify the efficacy of groenlandicine.Groenlandicine reduced the migration and proliferation while promoting apoptosis in OS cells, effectively damaging them. Meanwhile, groenlandicine exhibited weak cytotoxicity in 293T cells. Combination with cisplatin enhanced tumor-killing activity, markedly activating BAX, cleaved-Caspase-3, and cleaved-Caspase-9, while inhibiting the Bcl2 pathway in cisplatin-resistant OS cells. Moreover, the level of TOP1, elevated in cisplatin-resistant OS cells, was down-regulated by groenlandicine both in vitro and in vivo. Animal experiments confirmed that groenlandicine combined with cisplatin suppressed OS growth with lower nephrotoxicity.Groenlandicine induces apoptosis and enhances the sensitivity of drug-resistant OS cells to cisplatin via the BAX/Bcl-2/Caspase-9/Caspase-3 pathway. Groenlandicine inhibits OS cells growth by down-regulating TOP1 level.Therefore, groenlandicine holds promise as a potential agent for reversing cisplatin resistance in OS treatment.