Cell Behavior Research Articles

Effects of Capsaicin on Migration and Alkaline Phosphatase Activity of Dental Pulp Cells.

Dental pulp, a specialized mesenchymal tissue within teeth, is pivotal in dental health and tissue repair. Capsaicin, the primary pungent component of chili peppers, is known for its diverse pharmacological properties. While capsaicin's effects on various cell types have been studied, its impact on dental pulp cells remains relatively unexplored. This study investigated the influence of pure capsaicin extract on dental pulp cell behavior, focusing on cell viability, proliferation, migration, and alkaline phosphatase (ALP) activity. Capsaicin solution was prepared and diluted to various concentrations (1 nM, 0.01 µM, 0.1 µM, 1 µM, 10 µM, and 100 µM), then was tested on rat dental pulp cells (RPC-C2A). Cell viability and proliferation were assessed using the MTT assay. Boyden chamber tests and wound healing were used for evaluating cell migration. The activity of ALP was determined to show cell function during dental pulp repair. The data were analyzed using a one-way analysis of variance or an independent-sample Kruskal-Wallis, followed by multiple comparison tests. Capsaicin of 100 µM exhibited cytotoxicity, whereas those with lower concentrations stimulated cell proliferation. Wound healing assays revealed increased cell migration, particularly when cultured with 1 nM capsaicin (p = 0.002). Boyden chamber assays demonstrated enhanced cell invasion without statistical significance. ALP activity of dental pulp cells increased significantly at 1 nM (p < 0.001) and 1 µM (p = 0.021) capsaicin concentrations, indicating potential dentinogenesis and pulp repair. Capsaicin of lower concentrations, less than 10 µM, is likely to promote proliferation, migration, and ALP activity of dental pulp cells. Our findings offer potential applications for capsaicin as a medication for dental pulp repair.

Abstract PO4-28-03: Investigating the role of extracellular vesicles in tumor-stromal cell interactions in early-stage breast cancer invasion

Abstract Ductal carcinoma in situ (DCIS) is the most common form of early-stage breast cancer. Anywhere from 10%–30% of DCIS cases progress to invasive ductal carcinoma (IDC), resulting in poor prognosis and clinical challenges. Still, the cellular basis of this DCIS transition remains poorly understood. To escape the primary site during local invasion, DCIS cells navigate through the extracellular matrix and a diverse collection of stromal cells in the surrounding microenvironment. Cancer associated fibroblasts (CAFs) are a stromal cell population that can facilitate tumor invasion in part through extracellular matrix remodeling. CAFs at the primary tumor are largely derived from fibroblasts that have been reprogrammed to an activated state. In addition, several physical, cellular and metabolic changes within the tumor microenvironment (TME) are known to alter the breast cancer invasive capacity. Here we report that forced inhibition of cell invasion, by blocking the activity of known regulators of cell invasion in the ductal epithelial cell line, MCF10ADCIS.com, (herein referred to as DCIS.com), is accompanied by a marked reduction in extracellular vesicle (EV) secretion. Moreover, we show that DCIS.com cells conditioned with secreted EVs exhibit significantly enhanced invasive capacity relative to naïve DCIS.com cells. It is now well documented that breast and other tumor cells release heterogenous populations of EVs that can function as mediators of intercellular communication in the tumor microenvironment. EVs contain a host of bioactive cargo, such as membrane and cytosolic proteins, various RNA species including microRNAs, as well as dsDNA. These shed vesicles may deposit paracrine information and can also be taken up by stromal cells such as fibroblasts causing the recipient cells to undergo phenotypic changes that profoundly impact various facets of tumor progression. Conversely, EVs shed from stromal cells also affect tumor cell behavior. This unique form of intercellular crosstalk helps condition the TME and promotes both invasive and metastatic activity. In this regard, we found that incubation of human fibroblast cells with EVs shed from tumor cells leads to a loss of SMAD-7 expression (previously implicated in CAF reprogramming), and this loss is abrogated when shedding cells are depleted of miR-21 or regulators of miRNA trafficking to surface-derived EVs. Further, using spheroid invasion assays we demonstrate that fibroblast-derived EVs facilitate directed and leader-follower modes of migration, suggesting the possibility that CAFs likely deposit chemotactic cues to promote directional movement in DCIS. While the regulation of this behavior is still being explored, our observations suggest that EVs, in addition to facilitating distal proteolysis, may also support both autocrine and paracrine signaling during cell invasion. Finally, we are investigating how these pathways may also contribute to the ‘field effect’ of breast cancerization, a poorly understood process that introduces genetic and phenotypic changes in normal epithelia, potentially contributing to disease onset and/or recurrence. Citation Format: Madison Schmidtmann, Grace Richmond, Shireen Jayman, James Clancy, Crislyn D'Souza-Schorey. Investigating the role of extracellular vesicles in tumor-stromal cell interactions in early-stage breast cancer invasion [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-28-03.

Engineered Cell Membrane-Coated Nanoparticles: New Strategies in Glioma Targeted Therapy and Immune Modulation.

Gliomas, the most prevalent primary brain tumors, pose considerable challenges due to their heterogeneity, intricate tumor microenvironment (TME), and blood-brain barrier (BBB), which restrict the effectiveness of traditional treatments like surgery and chemotherapy. This review provides an overview of engineered cell membrane technologies in glioma therapy, with a specific emphasis on targeted drug delivery and modulation of the immune microenvironment. This study investigates the progress in engineered cell membranes, encompassing physical, chemical, and genetic alterations, to improve drug delivery across the BBB and effectively target gliomas. The examination focuses on the interaction of engineered cell membrane-coated nanoparticles (ECM-NPs) with the TME in gliomas, emphasizing their potential to modulate glioma cell behavior and TME to enhance therapeutic efficacy. The review further explores the involvement of ECM-NPs in immunomodulation techniques, highlighting their impact on immune reactions. While facing obstacles related to membrane stability and manufacturing scalability, the review outlines forthcoming research directions focused on enhancing membrane performance. This review underscores the promise of ECM-NPs in surpassing conventional therapeutic constraints, proposing novel approaches for efficacious glioma treatment.

Multiplexed Imaging Mass Cytometry Analysis Characterizes the Vascular Niche in Pancreatic Cancer.

Oncogenesis and progression of pancreatic ductal adenocarcinoma (PDAC) is driven by complex interactions between the neoplastic component and the tumor microenvironment (TME), which includes immune, stromal, and parenchymal cells. In particular, most PDACs are characterized by a hypovascular and hypoxic environment that alters tumor cell behavior and limits the efficacy of chemotherapy and immunotherapy. Characterization of the spatial features of the vascular niche could advance our understanding of inter- and intra-tumoral heterogeneity in PDAC. Here, we investigated the vascular microenvironment of PDAC by applying imaging mass cytometry using a 26-antibody panel on 35 regions of interest (ROIs) across 9 patients, capturing over 140,000 single cells. The approach distinguished major cell types, including multiple populations of lymphoid and myeloid cells, endocrine cells, ductal cells, stromal cells, and endothelial cells. Evaluation of cellular neighborhoods identified 10 distinct spatial domains, including multiple immune and tumor-enriched environments as well as the vascular niche. Focused analysis revealed differential interactions between immune populations and the vasculature and identified distinct spatial domains wherein tumor cell proliferation occurs. Importantly, the vascular niche was closely associated with a population of CD44-expressing macrophages enriched for a pro-angiogenic gene signature. Together, this study provides insights into the spatial heterogeneity of PDAC and suggests a role for CD44-expressing macrophages in shaping the vascular niche.

Chiral Arginine Modified Electrospun Membrane for Enhancing Tendon Healing

AbstractTendon injury is a common motor system disease, impairing joint mobility, and lowering quality of life. Once damaged, tendon has a limited capacity for regeneration. Clinically, available therapeutic strategies have not achieved satisfactory outcomes. Chiral biomaterials can effectively regulate cell behaviors and tissue healing, but have not been applied to injured tendon yet. Here, chiral arginine is attached to electrospun membrane to fabricate chiral scaffolds. L‐chiral scaffold, rather than D‐chiral or R‐chiral scaffold, promotes cell adhesion, proliferation, and tenogenic differentiation. The vinculin/FAK/YAP pathway is discovered to have a significant impact on the processes mentioned above. Additionally, L‐arginine efficiently eliminates reactive oxygen species (ROS) and generates nitric oxide (NO), safeguarding tendon stem/progenitor cells (TSPCs) against oxidative stress. The use of L‐chiral scaffold in a rat model of Achilles tendon injury increases the expression of markers related to tendons and the deposition of collagen. Moreover, L‐chiral scaffold improves tendon structural, functional, and mechanical properties. This L‐chiral scaffold comprehensively enhances tendon healing, providing a promising therapeutic strategy for tendon injury. As a simple and effective method, modification by chiral molecules enriches biomaterial functions and offers a novel option for tissue regeneration.

A Spontaneous In Situ Thiol-Ene Crosslinking Hydrogel with Thermo-Responsive Mechanical Properties.

The thermo-responsive behavior of Poly(N-isopropylacrylamide) makes it an ideal candidate to easily embed cells and allows the polymer mixture to be injected. However, P(NiPAAm) hydrogels possess minor mechanical properties. To increase the mechanical properties, a covalent bond is introduced into the P(NIPAAm) network through a biocompatible thiol-ene click-reaction by mixing two polymer solutions. Co-polymers with variable thiol or acrylate groups to thermo-responsive co-monomer ratios, ranging from 1% to 10%, were synthesized. Precise control of the crosslink density allowed customization of the hydrogel's mechanical properties to match different tissue stiffness levels. Increasing the temperature of the hydrogel above its transition temperature of 31 °C induced the formation of additional physical interactions. These additional interactions both further increased the stiffness of the material and impacted its relaxation behavior. The developed optimized hydrogels reach stiffnesses more than ten times higher compared to the state of the art using similar polymers. Furthermore, when adding cells to the precursor polymer solutions, homogeneous thermo-responsive hydrogels with good cell viability were created upon mixing. In future work, the influence of the mechanical micro-environment on the cell's behavior can be studied in vitro in a continuous manner by changing the incubation temperature.

AJAP1 Regulates Human Epithelial Cell Behaviors Required for Epicardium and EPDC Formation During Cardiac Development

Introduction: The epicardium forms the outermost layer of the heart. It is composed of a single layer of specialized epithelial cells that make important cellular contributions to cardiovascular development. During embryonic development, the epicardium is a source of multipotent progenitor cells, growth factors and extracellular matrix (ECM) components. The epicardium gives rise to multipotent epicardial-derived cells (EPDCs) which undergo epithelial-to-mesenchymal transition and differentiate into non-myocyte cardiac lineages. It is also a source of paracrine signals essential for myocardial growth and coronary vessel formation that become dormant after gestation. Adult epicardial cells can be reactivated in response to myocardial injury or ischemia to express a fetal gene program for regenerative cardiac repair. The drive to harness epicardial cells for cardiac regeneration is tethered to its roles in cardiovascular development. Thus, it is necessary to identify molecules that regulate epicardial cell behaviors required for epicardium formation and EPDC differentiation. Using our Tbx5-deficient mouse model of defective epicardium formation ( Tbx5epi−/−), we identified a transcript that is novel to the heart. Adherens junction associated protein 1 is a cell adhesion molecule that is encoded by the AJAP1 transcript which was downregulated in Tbx5epi−/− mice with impaired epicardium formation. Our preliminary studies revealed that AJAP is expressed in epithelial cells of the epicardium during heart development. Silencing of AJAP1 in primary cultured human epithelial cells reduces their ability to migrate. A role for AJAP1 in the epicardium during cardiac development remains to be identified. Study Objective: This study was designed to investigate the impact of AJAP1 on epithelial cell functions that are required for formation of the epicardium and differentiation of EPDCs. Methods: Studies were conducted in a primary human epithelial cell line, HMEpiC, which served as an in vitro model system to examine the contributions of AJAP1 to epithelial cell behaviors. HMEpiCs were transfected with small interfering RNAs (siRNAs) targeting either human AJAP1 to knock down its endogenous expression or a non-targeting negative control siRNA. AJAP1 knockdown was confirmed by quantitative PCR and immunofluorescence. We conducted assays on control and AJAP1 knockdown (KD) cells to examine the effects on epithelial cell behaviors contributing to epicardium formation and EPDC differentiation. This included analysis of cell proliferation, apoptosis, and adhesion to extracellular matrices. Results: Endogenous AJAP1 mRNA expression was reduced significantly by siRNA-mediated knockdown in human epithelial cells. We observed changes in cell morphology that correlated with a significant increase in apoptosis in AJAP1 KD cells compared to negative control cells. Proliferation of HMEpiCs was unchanged by AJAP1 knockdown. Also, we observed a significant increase in the adhesion of AJAP1 KD cells to certain extracellular matrices versus control cells. Conclusion: These findings provide evidence for the AJAP1 cell adhesion molecule to regulate epithelial cell behaviors that are crucial to formation of the epicardium and differentiation of EPDCs during cardiac development. PCOM Center for Chronic Disorders of Aging; PCOM Division of Research. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Noncanonical functions of telomerase and telomeres in viruses-associated cancer.

The cause of cancer is attributed to the uncontrolled growth and proliferation of cells resulting from genetic changes and alterations in cell behavior, a phenomenon known as epigenetics. Telomeres, protective caps on the ends of chromosomes, regulate both cellular aging and cancer formation. In most cancers, telomerase is upregulated, with the telomerase reverse transcriptase(TERT) enzyme and telomerase RNA component(TERC) RNA element contributing to the maintenance of telomere length. Additionally, it is noteworthy that two viruses, human papillomavirus (HPV) and Epstein-Barr virus (EBV), utilize telomerase for their replication or persistence in infected cells. Also, TERT and TERC may play major roles in cancer not related to telomere biology. They are involved in the regulation of gene expression, signal transduction pathways, cellular metabolism, or even immune response modulation. Furthermore, the crosstalk between TERT, TERC, RNA-binding proteins, and microRNAs contributes to a greater extent to cancer biology. To understand the multifaceted roles played by TERT and TERC in cancer and viral life cycles, and then to develop effective therapeutic strategies against these diseases, are fundamental for this goal. By investigating deeply, the complicated mechanisms and relationships between TERT and TERC, scientists will open the doors to new therapies. In its analysis, the review emphasizes the significance of gaining insight into the multifaceted roles that TERT and TERC play in cancer pathogenesis, as well as their involvement in the viral life cycle for designing effective anticancer therapy approaches.

Titanium alloys for orthopedic applications: A review on the osteointegration induced by physicomechanical stimuli

Titanium (Ti) alloys have been widely applied clinically due to their good biocompatibility, corrosion resistance and mechanical stability. However, how to improve the weak osteointegration caused by their intrinsic bio-inertness has been a long-standing puzzle. Osteogenic-related cells, endothelial cells (ECs) and macrophages are the main cells involved in osteointegration. In recent years, surface topography based on Ti alloys, which mimics the topographical cues of the extracellular matrix (ECM), has been confirmed to precisely regulate these cells' fate to improve osteointegration. More importantly, while serving as a physicomechanical stimuli, topographic surface modification is considered more biosafe and stable than biochemical cues. Nevertheless, how different topographic modifications affect cell behavior and the specific mechanisms involved remain elusive. In this review, we highlight the regulation of surface topography based on Ti alloys on the behaviors of bone repair-related cells, such as osteogenic-related cells, ECs and macrophages, and summarize the activated intracellular mechanotransduction pathways, which aims to provide basis and support for further research on osteointegration of Ti alloys.

Microfluidic chips for microbially induced calcium carbonate precipitation: Advantages, challenges, and insights

Microbially induced calcium carbonate precipitation (MICP) has garnered significant attention as a biomineralization process with diverse applications spanning from construction to environmental remediation. To propel MICP research and deepen our comprehension of MICP mechanisms, microfluidic chips have emerged as potent tools offering precise control over environmental parameters and real-time observations. Herein, we explore the benefits and challenges associated with employing microfluidic chips as a platform for investigating MICP. The advantages of microfluidic chips lie in their capacity to create controlled microenvironments conducive to emulating specific conditions crucial for MICP. The high-throughput nature of these devices accelerates experimentation by facilitating simultaneous testing of various microbial strains and nutrient compositions. Throughout the MICP process, observations were made on the behaviors of both bacterial cells and CaCO3 cementation. The inherent reduction in reagent consumption offered by microfluidics is both cost-effective and environmentally friendly. However, scaling up from microscale findings to practical applications necessitates careful consideration. Fully replicating the three-dimensional complexity and heterogeneous structures of the soil matrix, which influence microbial behavior, mineral distribution, and overall precipitation dynamics, using microfluidic chips remains challenging. Additionally, certain environmental complexities, including macroscopic soil components such as organic matter and various particle types, which significantly affect microbial activities and mineral precipitation patterns, may be difficult to replicate in microfluidic setups. However, microfluidic chips stand as invaluable tools for advancing MICP research. By addressing the advantages and disadvantages outlined here, researchers can harness the capabilities of microfluidic systems to unravel the intricacies of MICP, ultimately bridging the gap between fundamental understanding and real-world applications.

Modulating extracellular matrix stiffness: a strategic approach to boost cancer immunotherapy.

The interplay between extracellular matrix (ECM) stiffness and the tumor microenvironment is increasingly recognized as a critical factor in cancer progression and the efficacy of immunotherapy. This review comprehensively discusses the key factors regulating ECM remodeling, including the activation of cancer-associated fibroblasts and the accumulation and crosslinking of ECM proteins. Furthermore, it provides a detailed exploration of how ECM stiffness influences the behaviors of both tumor and immune cells. Significantly, the impact of ECM stiffness on the response to various immunotherapy strategies, such as immune checkpoint blockade, adoptive cell therapy, oncolytic virus therapy, and therapeutic cancer vaccines, is thoroughly examined. The review also addresses the challenges in translating research findings into clinical practice, highlighting the need for more precise biomaterials that accurately mimic the ECM and the development of novel therapeutic strategies. The insights offered aim to guide future research, with the potential to enhance the effectiveness of cancer immunotherapy modalities.

Cell-intrinsic and microenvironmental determinants of metastatic colonization.

Cancer metastasis is a biologically complex process that remains a major challenge in the oncology clinic, accounting for nearly all of the mortality associated with malignant neoplasms. To establish metastatic growths, carcinoma cells must disseminate from the primary tumour, survive in unfamiliar tissue microenvironments, re-activate programs of proliferation, and escape innate and adaptive immunosurveillance. The entire process is extremely inefficient and can occur over protracted timescales, yielding only a vanishingly small number of carcinoma cells that are able to complete all of the required steps. Here we review both the cancer-cell-intrinsic mechanisms and microenvironmental interactions that enable metastatic colonization. In particular, we highlight recent work on the behaviour of already-disseminated tumour cells, since meaningful progress in treating metastatic disease will clearly require a better understanding of the cells that spawn metastases, which generally have disseminated by the time of initial diagnosis.

MiR-122-5p regulates erastin-induced ferroptosis via CS in nasopharyngeal carcinoma

Nasopharyngeal carcinoma (NPC) is a tumor that occurs in the nasopharynx. Although advances in detection and treatment have improved the prognosis of NPC the treatment of advanced NPC remains challenging. Here, we explored the effect of microRNA (miR)-122-5p on erastin-induced ferroptosis in NPC cells and the role of ferroptosis in the development of NPC. The effect of miR-122-5p silencing and overexpression and the effect of citrate synthase on erastin-induced lipid peroxidation in NPC cells was analyzed by measuring the amounts of malondialdehyde, Fe2+, glutathione, and reactive oxygen species and the morphological alterations of mitochondria. The malignant biological behavior of NPC cells was examined by cell counting kit-8, EDU, colony formation, Transwell, and wound healing assays. The effects of miR-122-5p on cell proliferation and migration associated with ferroptosis were examined in vivo in a mouse model of NPC generated by subcutaneous injection of NPC cells. We found that erastin induced ferroptosis in NPC cells. miR-122-5p overexpression inhibited CS, thereby promoting erastin-induced ferroptosis in NPC cells and decreasing NPC cell proliferation, migration, and invasion.

Long non-coding RNA NEAT1 promotes aerobic glycolysis and progression of cervical cancer through WNT/β-catenin/PDK1 axis.

Cervical cancer is one of the most common gynecological cancers. Accumulated evidence shows that long non-coding RNAs (lncRNAs) play essential roles in cervical cancer occurrence and progression, but their specific functions and mechanisms remain to be further explored. The RT-qPCR assay was used to detect the expression of NEAT1 in cervical cancer tissues and cell lines. CCK-8, colony formation, flow cytometry, western blotting, and Transwell assays were used to evaluate the impact of NEAT1 on the malignant behavior of cervical cancer cells. Glucose consumption, lactate production, ATP levels, ROS levels, MMP levels, and the mRNA expressions of glycolysis-related genes and tricarboxylic acid cycle-related genes were detected to analyze the effect of NEAT1 on metabolism reprograming in cervical cancer cells. The expressions of PDK1, β-catenin and downstream molecules of the WNT/β-catenin signaling pathway in cervical cancer cells and tissues were detected by western blotting, RT-qPCR, immunofluorescence and immunohistochemistry assays. This study investigated the role and possible molecular mechanism of lncRNA nuclear paraspeckle assembly transcript 1 (NEAT1) in cervical cancer. Our results showed that NEAT1 was highly expressed in cervical cancer tissues and cell lines. Downregulation of NEAT1 inhibited the proliferation, migration, invasion and glycolysis of cervical cancer cells, while overexpression of NEAT1 led to the opposite effects. Mechanistically, NEAT1 upregulated pyruvate dehydrogenase kinase (PDK1) through the WNT/β-catenin signaling pathway, which enhanced glycolysis and then facilitated cervical cancer metastasis. Furthermore, NEAT1 maintained the protein stability of β-catenin but did not affect its mRNA level. We also excluded the direct binding of NEAT1 to the β-catenin protein via RNA pull-down assay. The suppressive impact of NEAT1 knockdown on cell proliferation, invasion, and migration was rescued by β-catenin overexpression. The WNT inhibitor iCRT3 attenuated the carcinogenic effect induced by NEAT1 overexpression. In summary, these findings indicated that NEAT1 may contribute to the progression of cervical cancer by activating the WNT/β-catenin/PDK1 signaling axis.

Oleic acid inhibits the migration and invasion of breast cancer cells with stemness characteristics through oxidative stress-mediated attenuation of the FAK/AKT/NF-κB pathway

Oleic acid, found in a variety of food sources, is beneficial to health. However, studies on its anticancer effects on cancer stem cells resistant to conventional treatments and on the mechanisms underlying such effects are scarce. Here, we evaluated the anticancer effects of oleic acid against breast cancer cells with stemness characteristics. Oleic acid inhibited the proliferation of breast cancer stem-like cells more significantly than that of their respective parental cells, and reduced the expression of stem cell markers and migration and invasion propensity. It also repressed the phosphorylation of FAK, AKT, and NF-κB, proteins involved in cell migration and invasion. Oleic acid-induced increase in reactive oxygen species levels mediated the inhibition of the FAK/AKT/NF-κB signaling axis. Our study demonstrates that oleic acid-induced oxidative stress plays an important role in inhibiting the metastatic behavior of breast cancer cells with stemness characteristics and is relevant for management of treatment-resistant breast cancer.

A Highly Efficient, Selective, and Thermally Stable Dielectric Multilayer Emitter for Solar Thermophotovoltaics

Herein, the design, optimization, fabrication, and characterization of a highly efficient selective emitter (SE) for solar thermophotovoltaic systems is presented. An SE consisting of three layers (SiNx–SiO2–TiO2) deposited on a tungsten substrate is optimized for use with photovoltaic (PV) cells based on III–V semiconductors, such as GaSb, InGaAs, and InGaAsSb. The fabricated SE shows an emitter efficiency (ηSE) of 50% when coupled with a PV cell having an energy bandgap of 0.63 eV. After thermal treatment carried out at 1000 °C for 8 h in a vacuum environment, ηSE of 46% is recorded, demonstrating the thermal stability of the proposed SE. Its behavior at high temperatures has also been studied using simulations based on the transfer matrix method and on refractive indices experimentally measured at different temperatures (up to 1000 °C). The results show ηSE of 44% in the energy bandgap range of 0.55–0.63 eV, proving that the proposed structure is promising and can operate at high temperatures. In addition, the behavior of a real PV cell is simulated, and calculations show a maximum PV cell efficiency of 15% at 1000 °C and 25% at 1600 °C, exceeding the Shockley–Queisser limit.

LncRNA LINC00847 accelerates melanoma progression by regulating miR-133a-3p/TGFBR1 axis.

Growing evidence has suggested that lncRNAs play a regulatory role in tumorigenesis. Dysregulation of a newly identified lncRNA (LINC00847) has been involved in several tumors. Nevertheless, the expression and roles of lncRNAs in skin melanoma remain unclear. Therefore, we attempted to investigate the expressions and roles of lncRNAs in this study. Expression levels of LINC00847 were quantified in tissue samples from the TCGA database and clinically recruited participants. LINC00847 was inhibited in cells by transfecting with si-LINC00847 or si-NC. Expressions of LINC00847 and miR-133a-3p were determined using RT-qPCR, and the TGFBR1 level was determined using Western blotting. Targeting sites of LINC00847 with miR-133a-3p and miR-133a-3p with TGFBR1 were predicted by bioinformatic tools and proved by dual-luciferase reporter system and RNA immunoprecipitation. Cell proliferation, invasion, and migration abilities were assessed using CCK8, cell colony formation, cell wound scratch, and transwell assay, respectively. In both TCGA and clinical cohorts, the expression of LINC00847 was abnormally upregulated in skin melanoma tissues than that of benign nevus. Besides, LINC00847 expression increased more markedly in A375 and SK-MEL-28 cells than in normal epidermal melanocytes (HEMa-LP cells). LINC00847 knockdown remarkably restrained skin melanoma cell proliferation, metastasis, and wound healing rate. Furthermore, miR-133a-3p/TGFBR1 was the downstream target for LINC00847. LINC00847 negatively regulated miR-133a-3p expression in skin melanoma cells. Both miR-133a-3p inhibitors and TGFBR1 vector transfection reversed the effect of LINC00847 silence in skin melanoma cells. LINC00847 was highly expressed in skin melanoma, and its overexpression accelerated the malignant tumor behavior of skin melanoma cells. The miR-133a-3p /TGFBR1 axis was involved in the roles of LINC00847 in skin melanoma.

EPAS1, a hypoxia‐ and ferroptosis‐related gene, promotes malignant behaviour of cervical cancer by ceRNA and super‐enhancer

AbstractHypoxia and Ferroptosis are associated with the malignant behaviour of cervical cancer. Endothelial PAS domain‐containing protein 1 (EPAS1) contributes to the progression of cervical cancer. EPAS1 plays important roles in hypoxia and ferroptosis. Using the GEO dataset, machine‐learning algorithms were used to screen for hypoxia‐ and ferroptosis‐related genes (HFRGs) in cervical cancer. EPAS1 was identified as the hub gene. qPCR and WB were used to investigate the expression of EPAS1 in normal and cervical cancer tissues. The proliferation, invasion and migration of EPAS1 cells in HeLa and SiHa cell lines were detected using CCK8, transwell and wound healing assays, respectively. Apoptosis was detected by flow cytometry. A dual‐luciferase assay was used to analyse the MALAT1‐miR‐182‐5P‐EPAS1 mRNA axis and core promoter elements of the super‐enhancer. EPAS1 was significantly overexpressed in cervical cancer tissues. EPAS1 could increase the proliferation, invasion, migration of HeLa and SiHa cells and reduce the apoptosis of HeLa and SiHa cell. According to the double‐luciferase assay, EPAS1 expression was regulated by the MALAT1‐Mir‐182‐5p‐EPAS1 mRNA axis. EPAS1 is associated with super‐enhancers. Double‐luciferase assay showed that the core elements of the super‐enhancer were E1 and E3. EPAS1, an HFRG, is significantly overexpressed in cervical cancer. EPAS1 promotes malignant behaviour of cervical cancer cells. EPAS1 expression is regulated by super‐enhancers and the MALAT1‐miR‐182‐5P‐ EPAS1 mRNA axis. EPAS1 may be a target for the diagnosis and treatment of cervical cancer.

Titanium dioxide nanotubes applied to conventional glass ionomer cement influence the expression of immunoinflammatory markers: An in vitro study

ObjectivesTo assess the impact of different concentrations TiO2-nt incorporated into a glass ionomer cement on the proliferation, mitochondrial metabolism, morphology, and pro- and anti-inflammatory cytokine production of cultured fibroblasts (NIH/3T3), whether or not stimulated by lipopolysaccharides (LPS-2 μg/mL, 24 h). MethodsTiO2-nt was added to KM (Ketac Molar EasyMix™, 3 %, 5 %, 7 % in weight); unblended KM was used as the control. The analyses included: Cell proliferation assay (n = 6; 24/48/72h); Mitochondrial metabolism assay (n = 6; 24/48/72h); Confocal laser microscopy (n = 3; 24/48/72h); Determination of biomarkers (IL-1β/IL-6/IL-10/VEGF/TNF) by using both multiplex technology (n = 6; 12/18 h) and the quantitative real-time PCR assay (q-PCR) (n = 3, 24/72/120 h). The data underwent analysis using both the Shapiro-Wilk and Levene tests, and by generalized linear models (α = 0.05). ResultsIt demonstrated that cell proliferation increased over time, regardless of the presence of TiO2-nt or LPS, and displayed a significant increase at 72 h; mitochondrial metabolism increased (p < 0.05), irrespective of exposure to LPS (p = 0.937); no cell morphology changes were observed; TiO2-nt reverted the impact of KM on the secreted levels of the evaluated proteins and the gene expressions in the presence of LPS (p < 0.0001). ConclusionsTiO2-nt did not adversely affect the biological behavior of fibroblastic cells cultured on GIC discs.

Construction of a Lentiviral Vector for Fgfr2 Overexpression and its Impact on the Biological Behavior of Cranial Suture Mesenchymal Stem Cells.

Suture mesenchymal stem cells (SuSCs), possessing self-renewal and multilineage differentiation abilities, play a crucial role in cranial bone growth. However, the impact of the disease-causing fibroblast growth factor receptor 2 (FGFR2) mutation on SuSCs in Crouzon syndrome has not been explored. This study aims to employ a lentivirus to overexpress Fgfr2 and investigate its role in the pathogenesis of Crouzon syndrome. Starting with the prevalent FGFR2 mutation site in patients with Crouzon syndrome, a lentiviral vector carrying the Fgfr2.C361Y mutation was developed and transfected into SuSCs, with a determined multiplicity of infection values. The experimental group, SuSCs+Fgfr2.C361Y, was compared with the empty vector and normal SuSC groups. Cell proliferation, cycle, apoptosis, and osteogenic functionality were assessed using CCK-8 assays, flow cytometry, ALP activity assays, and real-time quantitative polymerase chain reaction. The lentiviral vector effectively infected SuSCs, leading to heightened Fgfr2 expression, with optimal multiplicity of infection values of 80. The experimental group demonstrated decreased proliferation activity and a higher apoptosis rate compared with controls (P < 0.05). After osteogenic induction, the experimental group showed significantly higher ALP activity than controls (P < 0.05). Real-time quantitative polymerase chain reaction indicated lower mRNA expression levels of Gli1, Axin2, Pcna, Cdk2, and Bcl-2 in the experimental group than controls, whereas Bax, Runx2, and Bmp-2 showed higher expression (P < 0.05). This study constructed a lentivirus vector to upregulate Fgfr2 expression in SuSCs, suppressing stem cell stemness by inhibiting proliferation, promoting apoptosis, and accelerating premature osteogenic differentiation, resulting in premature suture closure. These findings establish the groundwork for further understanding the pathogenesis of Crouzon syndrome.