Cell Behavior Research Articles

ZC3H13-induced the m6A modification of hsa_circ_0081723 promotes cervical cancer progression via AMPK/p53 pathway.

N6-methyladenosine (m6A) modification and circular RNAs (circRNAs) have been confirmed to participate in cervical cancer (CC) progression. However, the function of a novel circRNA, hsa_circ_0081723, has not yet been explored in CC. Therefore, this study aimed to investigate the potential role of hsa_circ_0081723 and its m6A modification in CC. The hsa_circ_0081723 and ZC3H13 expressions were examined by qRT-PCR in the CC tissues, and their prognostic significance was evaluated via Kaplan-Meier Plotter. The role of hsa_circ_0081723 in CC progression was checked by loss-of-function assays. The relative protein levels of AMPK/p53 pathway were determined by western blotting. The interactions of hsa_circ_0081723 and ZC3H13 were verified via MeRIP and RNA stability assays. The hsa_circ_0081723 expression was elevated in CC samples, and its higher levels indicated high histological grade, high FIGO stage, poor differentiation, and poor prognosis. Functionally, silencing hsa_circ_0081723 impaired the malignant behavior of CC cells and enhanced the protein levels of key molecules of the AMPK signaling pathway. Moreover, ZC3H13 was also elevated in CC samples and demonstrated a positive association with hsa_circ_0081723. The relative enrichment of hsa_circ_0081723 m6A and its stability were enhanced in ZC3H13 overexpressed CC cells. Mechanically, ZC3H13 overexpression partially reversed the antitumor effects caused by hsa_circ_0081723 knockdown in CC cells. This study innovatively demonstrates that ZC3H13-mediated m6A modification of hsa_circ_0081723 promotes CC progression by modulating AMPK/p53 pathway. Our findings may contribute to the understanding of the molecular mechanisms underlying CC and offer potential therapeutic targets for clinical treatment.

Exploring the Role of Genetic and Environmental Features in Colorectal Cancer Development: An Agent-Based Approach.

The complexity of issues in cancer research has led to the introduction of powerful computational tools to help experimental in vivo and in vitro methods. These tools, which typically focus on studying cell behavior and dynamic cell populations, range from systems of differential equations that are solved numerically to lattice models and agent-based simulations. In particular, agent-based models (ABMs) are increasingly used due to their ability to incorporate multi-scale features, ranging from the individual to the population level. This approach allows for the combination of statistically aggregated assumptions with individual heterogeneity. In this work, we present an ABM that simulates tumor progression in a colonic crypt, to provide an experimental in silico environment for testing results achieved in traditional laboratory research and developing alternative scenarios of tumor development. The model also allows some speculations about causal relationships in biologically inspired systems.

ColMA‐based bioprinted 3D scaffold allowed to study tenogenic events in human tendon stem cells

AbstractThe advent of bioprinting has enabled the creation of precise three‐dimensional (3D) cell cultures suitable for biomimetic in vitro models. In this study, we developed a novel protocol for 3D printing methacrylated collagen (ColMa, or PhotoCol®) combined with tendon stem/progenitor cells (hTSPCs) derived from human tendon explants. Although pure ColMa has not previously been proposed as a printable hydrogel, this paper outlines a robust and highly reproducible pipeline for bioprinting this material. Indeed, we successfully fabricated a 3D bioengineered scaffold and cultured it for 21 days under perfusion conditions with medium supplemented with growth/differentiation factor‐5 (GDF‐5). This bioprinting pipeline and the culture conditions created an exceptionally favorable 3D environment, enabling the cells to proliferate, exhibit tenogenic behaviors, and produce a new collagen type I matrix, thereby remodeling the surrounding environment. Indeed, over the 21‐day culture period under perfusion condition, tenomodulin expression showed a significant upregulation on day 7, with a 2.3‐fold increase, compared to days 14 and 21. Collagen type I gene expression was upregulated nearly 10‐fold by day 14. This trend was further confirmed by western blot analysis, which revealed a statistically significant difference in tenomodulin expression between day 21 and both day 7 and day 14. For type I collagen, significant differences were observed between day 0 and day 21, as well as between day 0 and day 14, with a p‐value of 0.01. These results indicate a progressive over‐expression of type I collagen, reflecting cell differentiation towards a proper tenogenic phenotype. Cytokines, such as IL‐8 and IL‐6, levels peaked at 8566 and 7636 pg/mL, respectively, on day 7, before decreasing to 54 and 46 pg/mL by day 21. Overall, the data suggest that the novel ColMa bioprinting protocol effectively provided a conducive environment for the growth and proper differentiation of hTSPCs, showcasing its potential for studying cell behavior and tenogenic differentiation.

MiRNAs Involvement in Modulating Signalling Pathways Involved in Ros-Mediated Oxidative Stress in Melanoma.

Reactive oxygen species (ROS) are intermediates in oxidation-reduction reactions with the capacity to modify biomolecules and temporarily or permanently alter cell behaviour through signalling pathways under physiological and pathophysiological conditions where there is an imbalance between oxidative factors and the antioxidant response of the organism, a phenomenon known as oxidative stress. Evidence suggests that the differential modulation of ROS-mediated oxidative stress occurs in the pathogenesis and progression of melanoma, and that this imbalance in redox homeostasis appears to be functionally linked to microRNA (miRNA o miRs)-mediated non-mutational epigenetic reprogramming involving genes and transcription factors. The relationship between ROS-mediated stress control, tumour microenvironment, and miRNA expression in melanoma is not fully understood. The aim of this review is to analyse the involvement of miRNAs in the modulation of the signalling pathways involved in ROS-mediated oxidative stress in melanoma. It is hoped that these considerations will contribute to the understanding of the mechanisms associated with a potential epigenetic network regulation, where the modulation of oxidative stress is consolidated as a common factor in melanoma, and therefore, a potential footprint poorly documented.

Suspension Bead Loading (SBL): An Economical Protein Delivery Platform to Study URM1’s Behavior in Live Cells

AbstractUniquely modified synthetic proteins are difficult to produce in large quantities, which could limit their use in various in vitro settings and in cellular studies. In this study, we developed a method named “suspension bead loading” (SBL), to deliver protein molecules into suspended living cells using glass beads, which significantly reduces the amount of protein required for effective delivery. We investigated the delivery efficiency of functionally different proteins and evaluated the cytotoxic effect of our method and the chemical and functional integrity of the delivered protein. We utilized SBL to address questions related to ubiquitin‐related modifier 1 (URM1). Employing minimal protein quantities, SBL has enabled us to study its behavior within live cells under different redox conditions, including subcellular localization and conjugation patterns. We demonstrate that oxidative stress alters both the localization and conjugation pattern of URM1 in cells, highlighting its possible role in cellular response to such extreme conditions.

Suspension Bead Loading (SBL): An Economical Protein Delivery Platform to Study URM1's Behavior in Live Cells.

Uniquely modified synthetic proteins are difficult to produce in large quantities, which could limit their use in various in vitro settings and in cellular studies. In this study, we developed a method named "suspension bead loading" (SBL), to deliver protein molecules into suspended living cells using glass beads, which significantly reduces the amount of protein required for effective delivery. We investigated the delivery efficiency of functionally different proteins and evaluated the cytotoxic effect of our method and the chemical and functional integrity of the delivered protein. We utilized SBL to address questions related to ubiquitin-related modifier 1 (URM1). Employing minimal protein quantities, SBL has enabled us to study its behavior within live cells under different redox conditions, including subcellular localization and conjugation patterns. We demonstrate that oxidative stress alters both the localization and conjugation pattern of URM1 in cells, highlighting its possible role in cellular response to such extreme conditions.

Synergistic Anti-Tumor Effects of Newcastle Disease Virus and Doxorubicin: Evidence from A Murine Breast Cancer Model

BackgroundDespite the recent advances in the diagnosis and treatment of breast cancer, triple-negative breast cancer (TNBC) remains a clinical challenge due to its aggressive nature and resistance to conventional therapies. Virotherapy has emerged as a promising cancer treatment strategy, leveraging the ability of viruses to specifically target and replicate in cancerous cells. This study evaluated the oncolytic potential of a combined therapeutic strategy, utilizing Newcastle disease virus (NDV) and Doxorubicin hydrochloride (Dox) both in vitro and in vivo. MethodsThe in vitro experiments involved exposing human and mouse TNBC cell lines (MDA-MB-231 and 4T1, respectively) to NDV and Dox, individually or in combination. Cell viability assays and flow cytometry analyses were conducted to assess the synergistic effects of NDV and Dox on regulating breast cancer cell behavior in vitro. Furthermore, the immune-stimulating potential of NDV was investigated by examining its effects on dendritic cell (DC) maturation using flow cytometry and T cell proliferation. The in vitro anti-tumor effects of NDV were examined in both parental and tamoxifen-resistant cancer cells to assess its efficacy against chemoresistance. Animal models of breast cancer were treated with NDV in combination with Dox. The body weight changes, tumor volume, and survival rates of the mice were monitored throughout the study. Histopathological analyses were conducted to evaluate the potential toxic effects of the treatments. ResultsBased on the MTT results, NDV at optimal concentrations synergized the effect of Dox to reduce the viability of both MDA-MB231 and 4T1 cell lines (Isobologram combination index of less than 1). Additionally, individual treatment with NDV was able to significantly reduce the viability of patient-derived breast cancer cells, compared to the untreated control (P < 0.05) without affecting the cells of normal adjacent tissue. Furthermore, a combination of NDV and Dox significantly enhanced the percentage of early and late apoptotic cells in MDA-MB-231 (P < 0.0001) and late apoptotic cells in 4T1 (P < 0.0001), in comparison with individual treatment with these agents. Flow cytometry results showed that, compared to wild type MDA-MB-231 cells, NDV-infected MDA-MB-231 cells were better inducers of T cell proliferation and DC maturation as evidenced by increased proliferation index (P < 0.05) and elevated expression of CD1a, CD83, and CD86 (P < 0.0001), respectively. Moreover, co-treatment of both wild-type and (tamoxifen) TAM-resistant MCF-7/TAMR-1 cells with TAM and NDV significantly reduce the viability of the cancer cells (P < 0.0001). In tumor-bearing mice locally engrafted with 4T1 cells, combined treatment of NDV and Dox exhibited a marked reduction in median tumor volume compared to the control group, validating our in vitro findings on their synergistic anti-tumor effects. These findings suggest that combining NDV with Dox can effectively inhibit tumor progression and has the potential to reduce the dose, and consequently the toxic side-effects, of Dox in breast cancer therapy.

A programmable platform for probing cell migration and proliferation.

The advent of advanced robotic platforms and workflow automation tools has revolutionized the landscape of biological research, offering unprecedented levels of precision, reproducibility, and versatility in experimental design. In this work, we present an automated and modular workflow for exploring cell behavior in two-dimensional culture systems. By integrating the BioAssemblyBot® (BAB) robotic platform and the BioApps™ workflow automater with live-cell fluorescence microscopy, our workflow facilitates execution and analysis of in vitro migration and proliferation assays. Robotic assistance and automation allow for the precise and reproducible creation of highly customizable cell-free zones (CFZs), or wounds, in cell monolayers and "hands-free," schedulable integration with real-time monitoring systems for cellular dynamics. CFZs are designed as computer-aided design models and recreated in confluent cell layers by the BAB 3D-Bioprinting tool. The dynamics of migration and proliferation are evaluated in individual cells using live-cell fluorescence microscopy and an in-house pipeline for image processing and single-cell tracking. Our robotics-assisted approach outperforms manual scratch assays with enhanced reproducibility, adaptability, and precision. The incorporation of automation further facilitates increased flexibility in wound geometry and allows for many experimental conditions to be analyzed in parallel. Unlike traditional cell migration assays, our workflow offers an adjustable platform that can be tailored to a wide range of applications with high-throughput capability. The key features of this system, including its scalability, versatility, and the ability to maintain a high degree of experimental control, position it as a valuable tool for researchers across various disciplines.

Hyperlipidemia interferes with the behavior of variant NKT cells

Abstract Background NKT cells significantly influence atherosclerosis development. However, inconsistent findings on the function of the NKT cells and the invariant subset in atherosclerosis remains a challenge. Moreover, there is no clarity on the role and phenotype of the variant (vNKT) subset in atherosclerosis. Purpose To understand the role of the vNKT subsets in HFD-induced atherosclerosis. Methods We extracted the data for meta-analysis to understand the role of the whole NKT and iNKT cells in atherosclerosis. PRISMA guidelines were followed for the meta-analysis. In addition, C57BL/6 mice (WT) were fed with HFD for 20 weeks from 6-8 weeks of age. vNKTs were identified as CD3+NK1.1+CD1d-tetramer- within the CD3+NK1.1+ NKT cell population in both liver and spleen by flow cytometry. Intracellular cyotkines staining was performed. Statistical differences inthe means of two groups are calculated by two-tailed Student’s t-test with nonparametric Mann-Whitney tests. P-value&amp;lt;0.05 was considered significant. All data represented as mean±SEM. Results The NKT cell numbers increased significantly in the spleen, lymph nodes, blood, and liver in the HFD-fed mice fed from 1.5 to 24 weeks in Apoe-/-, Ldlr-/- in comparison to wild-type mice (SMD, 2.51 [95% CI, 1.42, 3.61]). Contrary to this, the iNKT cell numbers and the iNKT TCR gene expression levels, decreased significantly in the HFD-fed mice (SMD, -2.04 [95% CI, -3.34, -0.75]). The reduction in iNKT cell numbers is also supported by the clinical data from the AMI patients when compared to healthy controls [SMD = -1.81, 95% CI = -2.89, -0.74] (Figure 1). In HFD-fed C57BL/6 hyperlipidemic mice, significantly increased levels of total cholesterol (HFD vs Chow; 226.5±29.66 vs 118.5±10.56 mg/dL, n=4) and LDLc (44.56±11.96 vs 16.69±1.13 mg%, n=4) was observed. The number of vNKTs in the liver was significantly increased by 46% (HFD vs Chow 28.35±2.28 vs 19.41±1.36, n=6-7) (Figure 2A). However, the CD8+ vNKTs were significantly decreased by 68.6% in the liver (4.72±0.99 vs 15.06±0.96) and the CD4+ vNKTs decreased more than two times in the HFD-fed WT liver (7.39±0.76 vs 18.28±0.36) (Figure 2B). Also, the intracellular IFN-γ+ vNKT cells doubled in their numbers in the HFD-fed spleen (14.31±1.39 vs 6.367±0.87) compared to the chow-fed mice (Figure 2C). Conclusion(s) Meta-analysis revealed dual nature of the NKT cells and its invariant subsets in atherosclerosis, since the whole NKTs increase whereas the iNKTs decrease in number. The vNKT subset actively participates in immune modulation in the HFD-induced hyperlipidemia by increasing their number, as well as by differentially regulating CD8 and CD4 co-receptors between chow and HFD. HFD induced a pro-inflammatory nature in the splenic vNKTs but not in liver vNKTs, that in turn may aggravate other immune cells. This data confirms the dual nature of the NKT subsets and reveals a crucial systemic involvement of vNKT cells in the progression of dyslipidemia.

Toll-like receptor 2-mediated ERK activation significantly upregulates interleukin-6 expression in M2-polarized macrophages

Abstract Objectives M2-polarized macrophages and interleukin (IL)-6 significantly alter the tumor microenvironment and promote the malignant behaviors of tumor cells. This study aimed to establish M2-type macrophages from THP-1 cells, which are human leukemia monocytes, and investigate the significance of toll-like receptor 2 (TLR2) signaling in IL-6 production. Methods THP-1 cells were treated with phorbol 12-myristate 13-acetate, IL-4, and IL-13 to stimulate their differentiation into M2 macrophages. Cell differentiation was confirmed by cytokine production, marker expression, and morphological alterations. Treatment with TLR agonists induced TLR stimulation in M2 macrophages. Subsequently, secretion and expression levels of IL-6 in M2 macrophages were measured using enzyme-linked immunosorbent assay, quantitative reverse transcription-polymerase chain reaction, and western blotting. Results Myeloid differentiation factor 88, tumor necrosis factor-associated factor 6, and IL-1 receptor-associated kinase-1/4 signaling pathways contributed to IL-6 production upon TLR2 activation in M2 macrophages. While both TLR2 and TLR4 activated NF-κB in M2 macrophages, IL-6 production was mainly dependent on TLR2, not TLR4, suggesting the involvement of major mechanisms other than NF-κB in IL-6 production. Notably, TLR2-stimulated extracellular signal-regulated kinase (ERK) was necessary for abundant IL-6 production, indicating that TLR2-mediated ERK signaling plays an essential role in M2 macrophages. Conclusions These results highlight the significance of TLR2 signaling in IL-6 production by M2 macrophages and provide insights into the underlying regulatory mechanisms.

Biomaterials for Cell Manufacturing.

Cell therapies, potent populations of cells used to treat disease and injury, can be strategically manufactured with biomaterial intervention to improve clinical translation. In this viewpoint, we discuss biomaterial design and integration into cell manufacturing steps to achieve three main goals: scale-up, phenotype control, and selection of potent cells. Material properties can be engineered to influence the cell-biomaterial interface and, therefore, impart desirable cell behavior such as growth, secretory activity, and differentiation. Future directions for the field should capitalize on the combinatorial design of biomaterial properties to yield highly specific and potent cell populations. Furthermore, future biomaterials could contribute to novel high-throughput cell separation technologies that can individually select the most therapeutically relevant cells within a produced batch.

MECOM and the PRDM gene family in uterine endometrial cancer: bioinformatics and experimental insights into pathogenesis and therapeutic potentials

To elucidate the expression profiles, methylation states, and clinicopathological significance of the PRDM gene family, focusing on the MECOM gene's role in uterine endometrial cancer (UCEC) and its molecular interactions with the TGF-beta signaling pathway. Our methodology combined detailed bioinformatics analyses using UALCAN and GEPIA with in vitro assessments in HEC-1-A cells. Techniques included CRISPR-Cas9 for gene editing and various cellular assays (CCK-8, flow cytometry, Transwell) to evaluate the effects of MECOM on cell proliferation, migration, and apoptosis, alongside Western blot analysis for protein regulation in the TGF-beta pathway. MECOM was upregulated in UCEC tissues, influencing tumor cell behavior significantly. Knockout studies demonstrated reduced proliferation and migration and increased apoptosis, while overexpression showed reverse effects. Mechanistically, MECOM modulated critical proteins within the TGF-beta pathway, impacting cell cycle dynamics and apoptotic processes. The PRDM gene family, particularly MECOM, plays a crucial role in the pathogenesis and progression of UCEC, suggesting its utility as a target for novel therapeutic interventions. Our findings offer valuable insights for future research and potential clinical application in managing uterine endometrial cancer.

PIEZO1 mediates matrix stiffness-induced tumor progression in kidney renal clear cell carcinoma by activating the Ca2+/Calpain/YAP pathway

ObjectiveThe significance of physical factors in the onset and progression of tumors has been increasingly substantiated by a multitude of studies. The extracellular matrix, a pivotal component of the tumor microenvironment, has been the subject of extensive investigation in connection with the advancement of KIRC (Kidney Renal Clear Cell Carcinoma) in recent years. PIEZO1, a mechanosensitive ion channel, has been recognized as a modulator of diverse physiological processes. Nonetheless, the precise function of PIEZO1 as a transducer of mechanical stimuli in KIRC remains poorly elucidated. MethodsA bioinformatics analysis was conducted using data from The Cancer Genome Atlas (TCGA) and the Clinical Proteomic Tumor Analysis Consortium (CPTAC) to explore the correlation between matrix stiffness indicators, such as COL1A1 and LOX mRNA levels, and KIRC prognosis. Expression patterns of mechanosensitive ion channels, particularly PIEZO1, were examined. Collagen-coated polyacrylamide hydrogel models were utilized to simulate varying stiffness environments and study their effects on KIRC cell behavior in vitro. Functional experiments, including PIEZO1 knockdown and overexpression, were performed to investigate the molecular mechanisms underlying matrix stiffness-induced cellular changes. Interventions in the Ca2+/Calpain/YAP Pathway were conducted to evaluate their effects on cell growth, EMT, and stemness characteristics. ResultsOur findings indicate a significant correlation between matrix stiffness and the prognosis of KIRC patients. It is observed that higher mechanical stiffness can facilitate the growth and metastasis of KIRC cells. Notably, we have also observed that the deficiency of PIEZO1 hinders the proliferation, EMT, and stemness characteristics of KIRC cells induced by a stiff matrix. Our study suggests that PIEZO1 plays a crucial role in mediating KIRC growth and metastasis through the activation of the Ca2+/Calpain/YAP Pathway. ConclusionThis study elucidates a novel mechanism through which the activation of PIEZO1 leads to calcium influx, subsequent calpain activation, and YAP nuclear translocation, thereby contributing to the progression of KIRC driven by matrix stiffness.

Tuning porosity of macroporous hydrogels enables rapid rates of stress relaxation and promotes cell expansion and migration

Extracellular matrix (ECM) viscoelasticity broadly regulates cell behavior. While hydrogels can approximate the viscoelasticity of native ECM, it remains challenging to recapitulate the rapid stress relaxation observed in many tissues without limiting the mechanical stability of the hydrogel. Here, we develop macroporous alginate hydrogels that have an order of magnitude increase in the rate of stress relaxation as compared to bulk hydrogels. The increased rate of stress relaxation occurs across a wide range of polymer molecular weights (MWs), which enables the use of high MW polymer for improved mechanical stability of the hydrogel. The rate of stress relaxation in macroporous hydrogels depends on the volume fraction of pores and the concentration of bovine serum albumin, which is added to the hydrogels to stabilize the macroporous structure during gelation. Relative to cell spheroids encapsulated in bulk hydrogels, spheroids in macroporous hydrogels have a significantly larger area and smaller circularity because of increased cell migration. A computational model provides a framework for the relationship between the macroporous architecture and morphogenesis of encapsulated spheroids that is consistent with experimental observations. Taken together, these findings elucidate the relationship between macroporous hydrogel architecture and stress relaxation and help to inform the design of macroporous hydrogels for materials-based cell therapies.

Neurotrophic extracellular matrix proteins promote neuronal and iPSC astrocyte progenitor cell- and nano-scale process extension for neural repair applications.

The extracellular matrix plays a critical role in modulating cell behaviour in the developing and adult central nervous system influencing neural cell morphology, function and growth. Neurons and astrocytes, play vital roles in neural signalling and support respectively and respond to cues from the surrounding matrix environment. However, a better understanding of the impact of specific individual extracellular matrix proteins on both neurons and astrocytes is critical for advancing the development of matrix-based scaffolds for neural repair applications. This study aimed to provide an in-depth analysis of how different commonly used extracellular matrix proteins- laminin-1, Fn, collagen IV, and collagen I-affect the morphology and growth of trophic induced pluripotent stem cell (iPSC)-derived astrocyte progenitors and mouse motor neuron-like cells. Following a 7-day culture period, morphological assessments revealed that laminin-1, fibronectin, and collagen-IV, but not collagen I, promoted increased process extension and a stellate morphology in astrocytes, with collagen-IV yielding the greatest increases. Subsequent analysis of neurons grown on the different extracellular matrix proteins revealed a similar pattern with laminin-1, fibronectin, and collagen-IV supporting robust neurite outgrowth. fibronectin promoted the greatest increase in neurite extension, while collagen-I did not enhance neurite growth compared to poly-L-lysine controls. Super-resolution microscopy highlighted extracellular matrix-specific nanoscale changes in cytoskeletal organization, with distinct patterns of actin filament distribution where the three basement membrane-associated proteins (laminin-1, fibronectin, and collagen-IV) promoted the extension of fine cellular processes. Overall, this study demonstrates the potent effect of laminin-1, fibronectin and collagen-IV to promote both iPSC-derived astrocyte progenitor and neuronal growth, yielding detailed insights into the effect of extracellular matrix proteins on neural cell morphology at both the whole cell and nanoscale levels. The ability of laminin-1, collagen-IV and fibronectin to elicit strong growth-promoting effects highlight their suitability as optimal extracellular matrix proteins to incorporate into neurotrophic biomaterial scaffolds for the delivery of cell cargoes for neural repair.

Bioactive Microgels with Tunable Microenvironment as a 3D Platform to Guide the Complex Physiology of Hepatocellular Carcinoma Spheroids.

Miniaturized three-dimensional tissue models, such as spheroids, have become a highly useful and efficient platform to investigate tumor physiology and explore the effect of chemotherapeutic efficacy over traditional two-dimensional monolayer culture, since they can provide more in-depth analysis, especially in regards to intercellular interactions and diffusion. The development of most tumor spheroids relies on the high proliferative capacity and self-aggregation behavior of tumor cells. However, it often disregards the effect of microenvironmental factors mediated by extracellular matrix, which are indispensable components of tissue structure. In this study, hepatocellular carcinoma (HCC) cells are encapsulated in bioactive microgels consisting of gelatin and hyaluronic acid designed to emulate tumor microenvironment in order to induce hepatic tumor spheroid formation. Two different subtypes of HCC's, HepG2 and Hep3B cell lines, are explored. The physicomechanical and biochemical properties of the microgels, controlled by changing the crosslinking density and polymer composition, are clearly shown to have substantial influence over the formation and spheroid formation. Moreover, the spheroids made from different cells and microgel properties display highly variable chemoresistance effects, further highlighting the importance of microenvironmental factors guiding tumor spheroid physiology.

Piezoelectricity and flexoelectricity in biological cells: the role of cell structure and organelles.

Living tissues experience various external forces on cells, influencing their behaviour, physiology, shape, gene expression, and destiny through interactions with their environment. Despite much research done in this area, challenges remain in our better understanding of the behaviour of the cell in response to external stimuli, including the arrangement, quantity, and shape of organelles within the cell. This study explores the electromechanical behaviour of biological cells, including organelles like microtubules, mitochondria, nuclei, and cell membranes. A two-dimensional bio-electromechanical model for two distinct cell structures has been developed to analyze the behavior of the biological cell to the external electrical and mechanical responses. The piezoelectric and flexoelectric effects have been included via multiphysics coupling for the biological cell. All the governing equations have been discretized and solved by the finite element method. It is found that the longitudinal stress is absent and only the transverse stress plays a crucial role when the mechanical load is imposed on the top side of the cell through compressive displacement. The impact of flexoelectricity is elucidated by introducing a new parameter called the maximum electric potential ratio ( ). It has been found that depends upon the orientation angle and shape of the microtubules. The magnitude of exhibit huge change when we change the shape and orientation of the organelles, which in some cases (boundary condition (BC)-3) can reach to three times of regular shape organelles. Further, the study reveals that the number of microtubules significantly impacts effective elastic and piezoelectric coefficients, affecting cell behavior based on structure, microtubule orientation, and mechanical stress direction. The insight obtained from the current study can assist in advancements in medical therapies such as tissue engineering and regenerative medicine.

MiR-124-3p inhibits cell stemness in glioblastoma via targeting EPHA2 through ALKBH5-mediated m6A modification.

Glioblastoma (GBM) is the most aggressive form of glioma, characterized by high mortality and poor prognosis. Dysregulation of microRNAs (miRNAs) plays a critical role in the progression and metastasis of GBM. This study aimed to investigate the role and molecular mechanism of miR-124-3p in GBM. Levels of miR-124-3p, EPHA2, and ALKBH5 were measured using quantitative real-time polymerase chain reaction (qRT-PCR). Cell proliferation, migration, invasion, and stemness were assessed using the Cell Counting Kit-8 (CCK-8), colony formation, Transwell, and sphere formation assays, respectively. Bioinformatics prediction, dual-luciferase reporter assays, and RNA pull-down experiments were employed to validate the target of miR-124-3p. RNA binding protein immunoprecipitation (RIP) and methylated RNA immunoprecipitation (Me-RIP) were utilized to evaluate the regulation of miR-124-3p maturation by ALKBH5. The results indicated that overexpression of miR-124-3p inhibited the proliferation, migration, invasion, and stemness of GBM cells. EPHA2 was identified as a direct downstream target of miR-124-3p, and its overexpression reversed the inhibitory effects of miR-124-3p on cellular functions. Furthermore, miR-124-3p targeted EPHA2 to inactivate the Wnt/β-catenin pathway. Additionally, ALKBH5 negatively regulated miR-124-3p by impeding its processing. In conclusion, knockdown of ALKBH5 promoted the processing of pri-miR-124-3p, increasing mature miR-124-3p levels, which inhibited the malignant behaviors of GBM cells by targeting EPHA2. These findings highlight the importance of the ALKBH5/miR-124-3p/EPHA2 axis in GBM.

Dysregulated inclusion of BOLA3 exon 3 promoted by HNRNPC accelerates the progression of esophageal squamous cell carcinoma.

Dysregulated RNA splicing events produce transcripts that facilitate esophageal squamous cell carcinoma (ESCC) progression, but how this splicing process is abnormally regulated remains elusive. Here, we unveiled a novel alternative splicing axis of BOLA3 transcripts and its regulator HNRNPC in ESCC. The long-form BOLA3 (BOLA3-L) containing exon 3 exhibited high expression levels in ESCC and was associated with poor prognosis. Functional assays demonstrated the protumorigenic function of BOLA3-L in ESCC cells. Additionally, HNRNPC bound to BOLA3 mRNA and promoted BOLA3 exon 3 inclusion forming BOLA3-L. High HNRNPC expression was positively correlated with the presence of BOLA3-L and associated with an unfavorable prognosis. HNRNPC knockdown effectively suppressed the malignant biological behavior of ESCC cells, which were significantly rescued by BOLA3-L overexpression. Moreover, BOLA3-L played a significant role in mitochondrial structural and functional stability. E2F7 acted as a key transcription factor that promoted the upregulation of HNRNPC and inclusion of BOLA3 exon 3. Our findings provided novel insights into how alternative splicing contributes to ESCC progression.

Thermally Assisted Microfluidics to Produce Chemically Equivalent Microgels with Tunable Network Morphologies.

Although micron-sized microgels have become important building blocks in regenerative materials, offering decisive interactions with living matter, their chemical composition mostly significantly varies when their network morphology is tuned. Since cell behavior is simultaneously affected by the physical, chemical, and structural properties of the gel network, microgels with variable morphology but chemical equivalence are of interest. This work describes a new method to produce thermoresponsive microgels with defined mechanical properties, surface morphologies, and volume phase transition temperatures. A wide variety of microgels is synthesized by crosslinking monomers or star polymers at different temperatures using thermally assisted microfluidics. The diversification of microgels with different network structures and morphologies but of chemical equivalence offers a new platform of microgel building blocks with the ability to undergo phase transition at physiological temperatures. The method holds high potential to create soft and dynamic materials while maintaining the chemical composition for a wide variety of applications in biomedicine.