Analytical Cellular Pathology最新文献

Background: Fibroblasts play a crucial role in diabetic wound healing, and their senescence is the cause of delayed wound repair. It was reported that fibroblasts can secrete exosomes that can mediate a vital role in diabetic complications. Our purpose is to examine the biological function of high glucose (HG)-induced senescent fibroblasts from the perspective of exosomes and reveal the mechanism at cellular and animal levels. Methods: HG-induced senescent fibroblasts were measured by senescence-associated β-galactosidase staining and immunofluorescence. Flow cytometry, 5-ethynyl-2'-deoxyuridine (edu), and cell counting kit 8 (CCK-8) assay were applied to detect apoptosis and cell viability. Fibroblasts and endothelial cells were cocultured, and the migration and angiogenesis abilities were detected by scratch, transwell, and tube formation assays. Exosomes were isolated and identified from fibroblasts that were treated differently. Then, the function of exosomes was investigated in cells and mice, including examining the cellular phenotype changes, detecting the autophagy levels, and evaluating the wound healing rate. Furthermore, the potential mechanism by which senescent fibroblast-derived exosomes inhibit wound healing was examined via bioinformatics, real-time quantitive polymerase chain reaction (qPCR), transfection, and dual-luciferase assays. Results: It illustrated that HG-induced senescent fibroblasts exhibited adverse impacts on cellular proliferation, migration, and angiogenesis of endothelial cells via secreting exosomes, and senescent fibroblast-derived exosomes (S-Exos) can delay skin wound defects in mice. Subsequent differential analysis of the GSE153214 and GSE48417 datasets elucidated that miR-497 was the biomarker in the senescent fibroblasts. Interestingly, the miR-497 levels were also elevated in S-Exos. Its overexpression can regulate human umbilical vein endothelial cell function by regulating autophagy via targeting ATG13. Furthermore, in vivo experiments also illustrated that miR-497 can delay wound healing and reduce autophagy. Conclusions: Our study demonstrated that exosomes from senescent fibroblasts can impair endothelial cell function and impede diabetic wound healing. The underlying mechanism was that fibroblast-derived exosomal miR-497 can target ATG13 to reduce autophagy, offering insight into new therapy for diabetic complications and other diseases.

Background: Circular RNAs (circRNAs), covalently closed single-stranded RNAs, have been implicated in cancer progression. A previous investigation revealed that circ-ZEB1 is expressed abnormally in liver cancer. However, the roles of circ-ZEB1 in non-small cell lung cancer (NSCLC) are unknown. Methods: In this study, we used fluorescence in situ hybridization (FISH) and RT-qPCR to study circ-ZEB1 expression in NSCLC cells and tissues. A luciferase reporter assay was performed to validate downstream targets of circ-ZEB1. Transwell migration, 5-ethynyl-20-deoxyuridine (EdU), and cell counting kit-8 (CCK8) assays were performed to assess proliferation and migration. In vivo metastasis and tumorigenesis assays were also performed to investigate circ-ZEB1 functions during NSCLC. Results: Our results showed that circ-ZEB1 expression was increased in NSCLC tissues and cells. circ-ZEB1 downregulation suppressed NSCLC cell proliferation as well as migration in vitro and in vivo. Luciferase data confirmed EIF5A and miR-491-5p as downstream targets of circ-ZEB1. EIF5A overexpression and miR-491-5p suppression reversed NSCLC cell migration post circ-ZEB1 silencing. Conclusion: Our collective findings advised that circ-ZEB1 takes part in the malignant progression through regulating the miR-491-5p/EIF5A axis, highlighting its potential as an effective NSCLC therapeutic target.

Background: This study aims to study how gold nanoparticles (AuNPs) function in the recruitment and polarization of tumor-associated macrophages (TAMs) in hormone-sensitive prostate cancer (HSPC) and castration-resistant prostate cancer (CRPC). Methods: Phorbol ester (PMA)-treated THP-1 cells were cocultured with LNCaP or PC3 cells to simulate TAMs. Macrophage M2 polarization levels were detected using flow cytometry and M2 marker determination. ATG5 expression was detected by western blotting. Luciferase reporter assay was used to analyze the N6-methyladenosine (m⁶A) site activity of ATG5 3' untranslated regions (3'-UTRs). Methylated RNA immune precipitation (MeRIP)-quantitative polymerase chain reaction (qPCR) was performed to determine the m⁶A levels at ATG5 3'-UTR. Xenograft mouse models were used to determine the function of AuNPs in vivo. Results: Macrophages exhibited reduced M2 polarization in both HSPC and CRPC cells after AuNP treatment which was prevented by induction of autophagy. AuNP treatment decreased the m⁶A levels in the 3'-UTR of ATG5. Mutational analysis of potential m⁶A sites within ATG5 3'-UTR revealed that these sites were required for AuNP regulation, indicating that AuNPs inhibited ATG5 levels in an m⁶A-dependent manner. The mouse model revealed that AuNPs significantly reduced the M2 polarization of TAMs in an autophagy-dependent manner in vivo. This suggests that AuNPs inhibit tumor growth in vivo partially through targeting M2 TAM. Conclusion: The ATG5/autophagy pathway is inhibited by AuNP treatment in an METTL3/m⁶A-dependent manner. AuNPs inhibit the TAM M2 polarization in HSPC and CRPC by inhibiting ATG5/autophagy.

Shikonin is a plant medicine extracted from Lithospermum, which dominate influential antioxidant and antitumor effect. Here, we report that shikonin was capable of inducing human esophageal cancer EC9706 cell apoptosis and autophagy, in a time- and dose-dependent manner. Shikonin exposure repressed cell viability and migration and invasion capabilities and caused EC9706 cell autophagy and apoptosis by activating the AMPK/mTOR/ULK axis. Autophagy inhibition secured EC9706 cells against shikonin-induced autophagy and apoptosis and reversed the upregulation of AMPK and ULK phosphorylation and downregulation of mTOR phosphorylation provoked by shikonin. In summary, shikonin instigates EC9706 cell apoptosis and autophagy using the target AMPK/mTOR/ULK signal pathway axis, which provides a potential new target to treat human esophageal cancer.

Colorectal cancer (CRC) stands as a significant global health issue, marked by elevated occurrence and mortality statistics. Despite the availability of various treatments, including chemotherapy, radiotherapy, and targeted therapy, CRC cells often exhibit resistance to these interventions. As a result, it is imperative to identify the disease at an earlier stage and enhance the response to treatment by acquiring a deeper comprehension of the processes driving tumor formation, aggressiveness, metastasis, and resistance to therapy. The Hippo pathway plays a critical role in facilitating the initiation of tumorigenesis and frequently experiences disruption within CRC because of genetic mutations and modified expression in its fundamental constituents. Targeting upstream regulators or core Hippo pathway components may provide innovative therapeutic strategies for modulating Hippo signaling dysfunction in CRC. To advance novel therapeutic techniques for CRC, it is imperative to grasp the involvement of the Hippo pathway in CRC and its interaction with alternate signaling pathways, noncoding RNAs, gut microbiota, and the immune microenvironment. This review seeks to illuminate the function and control of the Hippo pathway in CRC, ultimately aiming to unearth innovative therapeutic methodologies for addressing this ailment.

Tumor microenvironment (TME) is essential for the development and progression of hepatocellular carcinoma (HCC). Exosomes participate in constructing TME by passing biological information, but the regulatory effect of PDL1 in exosomes on anticancer activity of CD8⁺ T cells in HCC still needs to be further explored. In this study, high level of PDL1 was found in plasma exosomes of HCC patients, which turned out to be significantly associated with the increased number of tumor nodules, the upregulated level of serum AFP, the raised tendency of TNM stage, and the poor prognosis of HCC. The expression of CD8 may be inhibited in HCC that is characterized with high level of PDL1, and the protein level of exosomal PDL1 was determined by intracellular PDL1 abundance. High level of exosomal PDL1 inhibited the proliferation and activation of CD8⁺ T cells, but exhibited limited effect on the proliferation of hepatic cancer cells. Moreover, the growth of tumors formed by hepatic cancer cells Hepa1-6 in C57L mice was significantly promoted by the exosomal PDL1, which might be caused by the inhibitory effect of exosomal PDL1 on CD8⁺ T cells. Thus, exosomal PDL1 promotes the development and progression of HCC through inhibiting the anticancer activity of CD8⁺ T cells. This study provides sights for understanding the oncogenic role of PDL1 and a reasonable explanation for the low efficacy of anti-PD1/PDL1 immunotherapies in HCC.

Background: As an important downstream effector of various signaling pathways, mTOR plays critical roles in regulating many physiological processes including erythropoiesis. It is composed of two distinct complexes, mTORC1 and mTORC2, which differ in their components and downstream signaling effects. Our previous study revealed that the inhibition of mTORC1 by rapamycin significantly repressed the erythroid progenitor expansion in the early stage but promoted enucleation and mitochondria clearance in the late stage of erythroid differentiation. However, the particular roles and differences of mTORC1 and mTORC2 in the regulation of erythropoiesis still remain largely unknown. In the present study, we investigated the comparative effects of dual mTORC1/mTORC2 mTOR kinase inhibitor AZD8055 and mTORC1 inhibitor rapamycin on erythroid differentiation in K562 cells induced by hemin and erythropoiesis in β-thalassemia mouse model. Materials and Methods: In vitro erythroid differentiation model of hemin-induced K562 cells and β-thalassemia mouse model were treated with AZD8055 and rapamycin. Cell Counting Kit-8 was used to detect cell viability. The cell proliferation, cell cycle, erythroid surface marker expression, mitochondrial content, and membrane potential were determined and analyzed by flow cytometry and laser scanning confocal microscopy. Globin gene expression during erythroid differentiation was measured by RT-qPCR. The mTORC2/mTORC1 and autophagy pathway was evaluated using western blotting. Results: Both AZD8055 and rapamycin treatments increased the expression levels of the erythroid differentiation-specific markers, CD235a, α-globin, γ-globin, and ε-globin. Notably, AZD8055 suppressed the cell proliferation and promoted the mitochondrial clearance of hemin-induced K562 cells more effectively than rapamycin. In a mouse model of β-thalassemia, both rapamycin and AZD8055 remarkably improve erythroid cell maturation and anemia. Moreover, AZD8055 and rapamycin treatment inhibited the mTORC1 pathway and enhanced autophagy, whereas AZD8055 enhanced autophagy more effectively than rapamycin. Indeed, AZD8055 treatment inhibited both mTORC2 and mTORC1 pathway in hemin-induced K562 cells. Conclusion: AZD8055 is more effective than rapamycin in inhibiting proliferation and promoting mitochondrial clearance in erythroid differentiation, which might provide us one more therapeutic option other than rapamycin for ineffective erythropoiesis treatment in the future. These findings also provide some preliminary information indicating the roles of mTORC1 and mTORC2 in erythropoiesis, and further studies are necessary to dissect the underlying mechanisms.

Background: Oral squamous cell carcinoma (OSCC) is a prevalent and aggressive form of head and neck cancer, often diagnosed at advanced stages. Elucidating the molecular mechanisms involved in the malignant transformation from normal oral tissue to oral preinvasive lesions (OPL) and primary OSCC could facilitate early diagnosis and improve therapeutic strategies. Methods: Differentially expressed genes (DEGs) were identified from the GSE30784 dataset by comparing normal oral tissue, oral dysplasia, and primary OSCC samples. Cross-validation was performed using an independent RNA-seq dataset, GSE186775. Protein-protein interaction (PPI) network analysis, gene ontology annotation, and pathway enrichment analysis were conducted on the common DEGs. Hub genes were identified, and their prognostic significance was evaluated using survival analysis. Transcription factor (TF) enrichment analysis, cross-validation, and immunohistochemistry analyses were also performed. Results: A total of 226 proteins and 677 interactions were identified in the PPI network, with 34 hub genes, including FN1, SERPINE1, PLAUR, THBS1, and ITGA6. Pathways such as "Formation of the cornified envelope," "Keratinization," and "Developmental biology" were enriched. Overexpression of SERPINE1, PLAUR, THBS1, and ITGA6 correlated with poor prognosis, while upregulation of CALML5 and SPINK5 was associated with favorable outcomes. NFIB emerged as the most significant TF-regulating hub genes. Immunohistochemistry validated ITGA6 overexpression in primary OSCC. Cross-validation using the RNA-seq dataset supported the involvement of critical genes in the malignant transformation process. Conclusion: This study identified vital genes, pathways, and prognostic markers involved in the malignant transformation from normal oral tissue to OPL and primary OSCC, providing insights for early diagnosis and targeted therapy development. Cross-validation with an independent RNA-seq dataset and immunohistochemistry reinforced the findings, supporting the robustness of the identified molecular signatures.

Background: Gastric cancer (GC) is the most common malignant tumor and ranks third in the world. LncRNA H19 (H19), one of the members of lncRNA, is overexpressed in various tumors. However, many undetermined molecular mechanisms by which H19 promotes GC progression still need to be further investigated. Methodology. A series of experiments was used to confirm the undetermined molecular mechanism including wound healing and transwell assays. Key Results. In this study, a significant upregulation of H19 expression was detected in GC cells and tissues. The poor overall survival was observed in GC patient with high H19 expression. Overexpression of H19 promoted the migration of GC cells, while knockdown of H19 significantly inhibited cell migration. Moreover, miR-148a-3p had a certain negative correlation with H19. Luciferase reporter assay confirmed that H19 could directly bind to miR-148a-3p. As expected, miR-148a mimics inhibited cell migration and invasion induced by H19 overexpression. The above findings proved that H19 functions as a miRNA sponge and verified that miR-148a-3p is the H19-associated miRNA in GC. We also confirmed that SOX-12 expression was upregulated in GC patient's samples. SOX-12 expression was positively correlated with expression of H19 and was able to directly bind to miR-148a-3p. Importantly, in vitro wound healing assay showed that knockout of SOX-12 could reverse the promoting effect of H19 overexpression on cell migration.

Conclusion: In conclusion, H19 has certain application value in the diagnosis and prognosis of GC. Specifically, H19 accelerates GCs to migration and metastasis by miR-138a-3p/SOX-12 axis.

Shock wave therapy (SWT) is a new alternative therapy for patients with severe coronary artery disease that improves myocardial ischemic symptoms by delivering low-energy shock wave stimulation to ischaemic myocardium with low-energy pulsed waves. However, the specific mechanism of its protective effect is not fully understood, especially for the protective mechanism in cardiomyocytes after hypoxia/reoxygenation (H/R). We selected a rat H9c2 cardiomyocyte cell line to establish a stable H/R cardiomyocyte injury model by hypoxia/reoxygenation, and then used SWT for therapeutic intervention to explore its cardiomyocyte protective mechanisms. The results showed that SWT significantly increased cell viability and GSH levels while decreasing LDH levels, ROS levels, and MDA levels. SWT also improved mitochondrial morphology and function of cells after H/R. Meanwhile, we found that SWT could increase the expression of GPX4, xCT, and Bcl-2, while decreasing the expression of Bax and cleaved caspase-3, and inhibiting cardiomyocyte apoptosis and ferroptosis. Moreover, this protective effect of SWT on cardiomyocytes could be significantly reversed by knockdown of xCT, a key regulator protein of ferroptosis. In conclusion, our study shows that SWT can attenuate hypoxia-reoxygenation-induced myocardial injury and protect cardiomyocyte function by inhibiting H/R-induced apoptosis and ferroptosis, and this therapy may have important applications in the treatment of clinical myocardial ischemic diseases.