Osteocyte-derived exosomes regulate the DLX2/wnt pathway to alleviate osteoarthritis by mediating cartilage repair.

BACKGROUND: Chondrocyte viability, apoptosis, and migration are closely related to cartilage injury in osteoarthritis (OA) joints. Exosomes are identified as potential therapeutic agents for OA. OBJECTIVE: This study aimed to investigate the role of exosomes derived from osteocytes in OA, particularly focusing on their effects on cartilage repair and molecular mechanisms. METHODS: An injury cell model was established by treating chondrocytes with IL-1ß. Cartilage repair was evaluated using cell counting kit-8, flow cytometry, scratch test, and Western Blot. Molecular mechanisms were analyzed using quantitative real-time PCR, bioinformatic analysis, and Western Blot. An OA mouse model was established to explore the role of exosomal DLX2 in vivo. RESULTS: Osteocyte-released exosomes promoted cell viability and migration, and inhibited apoptosis and extracellular matrix (ECM) deposition. Moreover, exosomes upregulated DLX2 expression, and knockdown of DLX2 activated the Wnt pathway. Additionally, exosomes attenuated OA in mice by transmitting DLX2. CONCLUSION: Osteocyte-derived exosomal DLX2 alleviated IL-1ß-induced cartilage repair and inactivated the Wnt pathway, thereby alleviating OA progression. The findings suggested that osteocyte-derived exosomes may hold promise as a treatment for OA.

ROR2 promotes invasion and chemoresistance of triple-negative breast cancer cells by activating PI3K/AKT/mTOR signaling.

Objective: This study aimed to investigate the role of receptor tyrosine kinase-like orphan receptor 2 (ROR2) in triple-negative breast cancer (TNBC). Methods: ROR2 expression in primary TNBC and metastatic TNBC tissues was analyzed by immunohistochemical staining and PCR. ROR2 expression in TNBC cell lines was detected by PCR and Western blot analysis. The migration, invasion and chemosensitivity of TNBC cells with overexpression or knockdown of ROR2 were examined. Results: ROR2 expression was high in metastatic TNBC tissues. ROR2 knockdown suppressed the migration, invasion and chemoresistance of TNBC cells. ROR2 overexpression in MDA-MB-435 cells promoted the migration, invasion, and chemoresistance. Moreover, ROR2 knockdown in HC1599 and MDA-MB-435 adriamycin-resistant cells enhanced chemosensitivity to adriamycin. ROR2 could activate PI3K/AKT/mTOR signaling in TNBC cells. Conclusion: ROR2 is upregulated and promotes metastatic phenotypes of TNBC by activating PI3K/AKT/mTOR signaling.

LncRNA PCGEM1 facilitates cervical cancer progression via miR-642a-5p/KIF5B axis.

At present, the role of many long non-coding RNAs (lncRNAs) as tumor suppressors in the formation and development of cervical cancer (CC) has been studied. However, lncRNA prostate cancer gene expression marker 1 (PCGEM1), whose high expression not only aggravates ovarian cancer but also can induce tumorigenesis and endometrial cancer progression, has not been studied in CC. The objective of this study was to investigate the expression and the underlying role of PCGEM1 in CC. The relative expression of PCGEM1 in CC cells was detected by real-time PCR. After the suppression of PCGEM1 expression by shRNA, the changes in the proliferation, migration, and invasion capacities were detected via CCK-8 assay, EdU assay, and colony formation assay wound healing assay. Transwell assay and the changes in expressions of epithelial-to-mesenchymal transition (EMT) markers were determined by western blot and immunofluorescence. The interplay among PCGEM1, miR-642a-5p, and kinesin family member 5B (KIF5B) was confirmed by bioinformatics analyses and luciferase reporter assay. Results showed that PCGEM1 expressions were up-regulated within CC cells. Cell viabilities, migration, and invasion were remarkably reduced after the suppression of PCGEM1 expression by shRNA in Hela and SiHa cells. N-cadherin was silenced, but E-cadherin expression was elevated by sh-PCGEM1. Moreover, by sponging miR-642a-5p in CC, PCGEM1 was verified as a competitive endogenous RNA (ceRNA) that modulates KIF5B levels. MiR-642a-5p down-regulation partially rescued sh-PCGEM1's inhibitory effects on cell proliferation, migration, invasion, and EMT process. In conclusion, the PCGEM1/miR-642a-5p/KIF5B signaling axis might be a novel therapeutic target in CC. This study provides a research basis and new direction for targeted therapy of CC.

IKIP downregulates THBS1/FAK signaling to suppress migration and invasion by glioblastoma cells.

Background: Inhibitor of NF-κB kinase-interacting protein (IKIP) is known to promote proliferation of glioblastoma (GBM) cells, but how it affects migration and invasion by those cells is unclear. Methods: We compared levels of IKIP between glioma tissues and normal brain tissue in clinical samples and public databases. We examined the effects of IKIP overexpression and knockdown on the migration and invasion of GBM using transwell and wound healing assays, and we compared the transcriptomes under these different conditions to identify the molecular mechanisms involved. Results: Based on data from our clinical samples and from public databases, IKIP was overexpressed in GBM tumors, and its expression level correlated inversely with survival. IKIP overexpression in GBM cells inhibited migration and invasion in transwell and wound healing assays, whereas IKIP knockdown exerted the opposite effects. IKIP overexpression in GBM cells that were injected into mouse brain promoted tumor growth but inhibited tumor invasion of surrounding tissue. The effects of IKIP were associated with downregulation of THBS1 mRNA and concomitant inhibition of THBS1/FAK signaling. Conclusions: IKIP inhibits THBS1/FAK signaling to suppress migration and invasion of GBM cells.

PRL1 and PRL3 promote macropinocytosis via its lipid phosphatase activity.

PRL1 and PRL3, members of the protein tyrosine phosphatase family, have been associated with cancer metastasis and poor prognosis. Despite extensive research on their protein phosphatase activity, their potential role as lipid phosphatases remains elusive. Methods: We conducted comprehensive investigations to elucidate the lipid phosphatase activity of PRL1 and PRL3 using a combination of cellular assays, biochemical analyses, and protein interactome profiling. Functional studies were performed to delineate the impact of PRL1/3 on macropinocytosis and its implications in cancer biology. Results: Our study has identified PRL1 and PRL3 as lipid phosphatases that interact with phosphoinositide (PIP) lipids, converting PI(3,4)P2 and PI(3,5)P2 into PI(3)P on the cellular membranes. These enzymatic activities of PRLs promote the formation of membrane ruffles, membrane blebbing and subsequent macropinocytosis, facilitating nutrient extraction, cell migration, and invasion, thereby contributing to tumor development. These enzymatic activities of PRLs promote the formation of membrane ruffles, membrane blebbing and subsequent macropinocytosis. Additionally, we found a correlation between PRL1/3 expression and glioma development, suggesting their involvement in glioma progression. Conclusions: Combining with the knowledge that PRLs have been identified to be involved in mTOR, EGFR and autophagy, here we concluded the physiological role of PRL1/3 in orchestrating the nutrient sensing, absorbing and recycling via regulating macropinocytosis through its lipid phosphatase activity. This mechanism could be exploited by tumor cells facing a nutrient-depleted microenvironment, highlighting the potential therapeutic significance of targeting PRL1/3-mediated macropinocytosis in cancer treatment.

PLAUR facilitates the progression of clear cell renal cell carcinoma by activating the PI3K/AKT/mTOR signaling pathway.

Background: PLAUR has been found upregulated in various tumors and closely correlated with the malignant phenotype of tumor cells. The aim of this study was to investigate the relationship between PLAUR and clear cell renal cell carcinoma (ccRCC) and its potential mechanism of promoting tumor progression. Methods: The expression levels and clinical significance of PLAUR, along with the associated signaling pathways, were extensively investigated in ccRCC samples obtained from The Cancer Genome Atlas (TCGA). PLAUR expression in 20 pairs of ccRCC tumor tissues and the adjacent tissues was assessed using qRT-PCR and IHC staining. Additionally, a series of in vitro experiments were conducted to investigate the impact of PLAUR suppression on cellular proliferation, migration, invasion, cell cycle progression, and apoptosis in ccRCC. The Western blot analysis was employed to investigate the expression levels of pivotal genes associated with the PI3K/AKT/mTOR signaling pathway. Results: The expression of PLAUR was significantly upregulated in ccRCC compared to normal renal tissues, and higher PLAUR expression in ccRCC was associated with a poorer prognosis than low expression. The in-vitro functional investigations demonstrated that knockdown of PLAUR significantly attenuated the proliferation, migration, and invasion capabilities of ccRCC cells. Concurrently, PLAUR knockdown effectively induced cellular apoptosis, modulated the cell cycle, inhibited the EMT process, and attenuated the activation of the PI3K/AKT/mTOR signaling pathway. PLAUR may represent a key mechanism underlying ccRCC progression. Conclusions: The involvement of PLAUR in ccRCC progression may be achieved through the activation of the PI3K/AKT/mTOR signaling pathway, making it a reliable biomarker for the identification and prediction of ccRCC.

[miR-342-3p Promotes the Proliferation, Migration, and Invasion of Clear Cell Renal Cell Carcinoma Cells by Targeted Inhibition of PPM1E]. / miR-342-3p通过靶向抑制PPM1E促进肾透明细胞癌细胞的增殖、迁移和侵袭.

Objective: To explore the effects of microRNA-342-3p/Mg2+Mn2+-dependent protein phosphatase 1E (miR-342-3p/PPM1E) on the proliferation, migration, and invasion of clear cell renal cell carcinoma (ccRCC) cells. Methods: The gene chips GSE12105, GSE23085, GSE66271, and GSE66270 were searched, and the relationship between miR-342-3p, PPM1E, and the clinical malignant phenotypes of ccRCC was analyzed. ACHN and 769-P cells were transfected with miR-342-3p inhibitor. The effects of miR-342-3p on cell proliferation, migration, and invasion were examined. ACHN cell line with stable and high expression of miR-342-3p was constructed, and the tumorigenicity of the cell line in BALB/c nude mice was observed. The targeted relationship between miR-342-3p and PPM1E was verified by dual-luciferase reporter gene assay. The cells were transfected with miR-342-3p mimic and pcDNA-PPM1E plasmids to observe whether PPM1E could reverse the effects of miR-342-3p overexpression on the proliferation, migration, and invasion of the cells. Results: The expression of miR-342-3p was upregulated in ccRCC, and there were significant differences among patients with tumors of different T stages and G stages and those with different prognoses (P<0.05). The overall survival in the miR-342-3p high-expression group was significantly shorter than that in the low-expression group (P<0.05). Compared with those in the miR-NC group, the miR-342-3p level was significantly downregulated in the inhibitor group, and the cell proliferation ability and the numbers of migrating and invading cells were also significantly decreased (P<0.05). Compared with the miR-NC group, miR-342-3p group had significantly increased volume and mass of tumor tissues and miR-342-3p level, but significantly decreased level of PPM1E mRNA (P<0.05). The expression of PPM1E was downregulated in ccRCC, and there were significant differences among patients with tumors of different M stages, N stages, and G stages, and different recurrence statuses (P<0.05). The miR-342-3p could inhibit the expression of PPM1E in a targeted way. Compared with the miR-NC group, the miR-342-3p group had significantly increased cell proliferation ability and increased numbers of migrating and invading cells (P<0.05). However, PPM1E could reverse the promotion effect of miR-342-3p mimic on ccRCC cells (P<0.05). Conclusion: The miR-342-3p can inhibit PPM1E expression in a targeted way, and thus promotes the proliferation, migration, and invasion of ccRCC cells.

[Relationship Between the Migration of Endogenous Neural Stem Cells and the Pattern of Change in Immune Cell Phenotypes in the Microenvironment After Intracerebral Hemorrhage in Rats]. / å¤§é¼ è„‘å‡ºè¡€åŽå† æºæ€§ç¥žç»å¹²ç»†èƒžè¿ç§»å’Œå¾®çŽ¯å¢ƒä¸­å ç–«ç»†èƒžè¡¨åž‹å˜åŒ–è§„å¾‹çš„å ³ç³».

Objective: Intracerebral hemorrhage (ICH), the second most common type of stroke, can cause long-lasting disability in the afflicted patients. The study was conducted to examine the patterns of change in endogenous neural stem cells (eNSCs) and in the regenerative microenvironment after ICH, to observe the relationship between the migration of eNSCs and the pattern of change in the polarization state of immune cells in the microenvironment, and provide a research basis for research on clinical nerve repair. Methods: The collagenase injection method was used for modeling. The ICH model was induced in adult female Sprague-Dawley (SD) rats by injecting type VII collagenase (2 U) into the brain tissue of rats. All the experimental rats weighed 280-300 g. In order to simulate the ICU at different time points, including the acute phase (within 1 week), subacute phase (1-3 weeks), and the chronic phase (over 3 weeks), brain tissues were harvested at 3 day post injection (3 DPI), 10 DPI, 20 DPI, and 30 DPI to evaluate the modeling effect. Immunofluorescence staining of the brain tissue sections was performed with DCX antibody to observe the pattern of change in the migration of eNSCs in the brain tissue at different time points. Immunofluorescence staining of brain tissue sections was performed with CD206 antibody and CD86 antibody for respective observation of the pattern of change in pro-inflammatory (M1-type) and anti-inflammatory (M2-type) immune cells in the regenerative microenvironment of the brain tissue after ICM. Results: Spontaneous ICH was successfully induced by injecting type Ⅶ collagenase into the brain tissue of SD rats. The volume of the hematoma formed started to gradually increase at 3 DPI and reached its maximum at 10 DPI. After that, the hematoma was gradually absorbed and was completely absorbed by 30 DPI. Analysis of the pattern of changes in eNSCs in the brain tissue showed that a small number of eNSCs were activated at 3 DPI, but very soon their number started to decrease. By 10 DPI, eNSCs gradually began to increase. A large number of eNSCs migrated to the hemorrhage site at 20 DPI. Then the number of eNSCs decreased significantly at 30 DPI (P<0.01). Analysis of the immune microenvironment of the brain tissue showed that pro-inflammatory (M1 type) immune cells increased significantly at 10 and 20 DPI (P<0.01) and decreased at 30 DPI. Anti-inflammatory (M2 type) immune cells began to increase gradually at 3 DPI, decreased significantly at 20 DPI (P<0.05), and then showed an increase at 30 DPI. Conclusion: After ICH in rats, eNSCs migrating toward the site of ICH first increase and then decrease. The immune microenvironment demonstrates a pattern of change in which inflammation is suppressed at first, then promoted, and finally suppressed again. Inflammation may have a stimulatory effect on the migration of eNSCs, but excessive inflammatory activation has an inhibitory effect on the differentiation and further activation of eNSCs. After ICH, the early stage of repair and protection (10 d) and the subacute phase (20 d) may provide the best opportunities for intervention.

USP18 promotes colon adenocarcinoma progression via targeting the ERK-MNK signaling pathway.

BACKGROUND: Colorectal cancer is the third most common malignancy worldwide and is one of the leading causes of cancer-related mortality. Ubiquitin-specific peptidase 18 (USP18) protein has been reported to exert different tumor-related effects in distinct tumor types. Here, we initially investigated the expression and signaling pathways of USP18 in colon adenocarcinoma (COAD). METHODS: A quantitative real-time PCR was conducted to evaluate the mRNA level of USP18 in cultured cells. Immunohistochemical staining was used to explore the protein expression of USP18 in clinical COAD samples. Specific knockdown was achieved by transient transfection of small interfering RNAs into SW480 and HT29 cells using Lipo3000. Cell conting kit-8 assay, transwell assay and matrigel-transwell assays were conducted to evaluate proliferation, migration and invasion capacities, respectively. Western blotting was performed to analyze downstream signaling pathways. A chi-squared test and univariate and multivariate analyses were used to evaluate the clinical data. Xenografts from mice model were assessed to validate the in vitro findings. RESULTS: Higher USP18 level was identified in COAD tissues and was positively correlated with advanced tumor stage. High USP18 protein expression indicated poorer prognosis of COAD patients. Silencing USP18 suppressed COAD cell proliferation and invasion via destabilizing extracellular signal-regulated kinase (ERK) protein and suppressing ERK downstream pathways. Simultaneously silencing interferon-stimulated gene 15 (ISG15) with USP18 can partially rescue the tumor cell viability, indicating its involvement in USP18 signaling. The oncogenic effects of USP18 were also confirmed in mice models. CONCLUSIONS: USP18 plays oncogenic effects in colon adenocarcinoma via ISG15-ERK pathways. High USP18 expression indicates poor clinical outcomes for colon adenocarcinoma patients.

An epidermal growth factor receptor-targeting immunotoxin based on IgG shows potent antitumor activity against head and neck cancer.

The epidermal growth factor receptor (EGFR) is an important target for cancer therapies. Many head and neck cancer (HNC) cells have been reported to overexpress EGFR; therefore, anti-EGFR therapies have been attempted in patients with HNC. However, its clinical efficacy is limited owing to the development of drug resistance. In this study, we developed an EGFR-targeting immunotoxin consisting of a clinically proven anti-EGFR IgG (cetuximab; CTX) and a toxin fragment (LR-LO10) derived from Pseudomonas exotoxin A (PE) using a novel site-specific conjugation technology (peptide-directed photo-crosslinking reaction), as an alternative option. The immunotoxin (CTX-LR-LO10) showed specific binding to EGFR and properties of a typical IgG, such as stability, interactions with receptors of immune cells, and pharmacokinetics, and inhibited protein synthesis via modification of elongation factor-2. Treatment of EGFR-positive HNC cells with the immunotoxin resulted in apoptotic cell death and the inhibition of cell migration and invasion. The efficacy of CTX-LR-LO10 was evaluated in xenograft mouse models, and the immunotoxin exhibited much stronger tumor suppression than CTX or LR-LO10. Transcriptome analyses revealed that the immunotoxins elicited immune responses and altered the expression of genes related to its mechanisms of action. These results support the notion that CTX-LR-LO10 may serve as a new therapeutic agent targeting EGFR-positive cancers.

Resistance to gemcitabine is mediated by the circ_0036627/miR-145/S100A16 axis in pancreatic cancer.

The development of gemcitabine (GEM) resistance severely limits the treatment efficacy in pancreatic cancer (PC) and increasing evidence highlights the vital roles of circular RNAs (circRNAs) in the tumorigenesis, progression and drug resistance of PC. However, the circRNAs underlying GEM resistance development of PC remains to be clarified. The current research aims to unveil the roles of circ_0036627 in dictating the aggressiveness and GEM sensitivity in PC. We reported the increased expression of circ_0036627 in PC tissues and PC cell lines. Elevated circ_0036627 expression level was correlated with advanced tumour grade and poor overall survival in PC patients. Functional assays and in vivo experiments demonstrated that circ_0036627 overexpression was required for the proliferation, migration invasion and GEM resistance in PC cells. circ_0036627 knockdown suppressed tumour development in vivo. The molecular analysis further showed that circ_0036627 increased S100A16 expression by sponging microRNA-145 (miR-145), a tumour-suppressive miRNA that could significantly attenuate PC cell proliferation, migration, invasion and GEM resistance. Furthermore, our findings suggested that S100A16 acted as an oncogenic factor to promote aggressiveness and GEM resistance in PC cells. In conclusion, the current findings provide new mechanistic insights into PC aggressiveness and GEM resistance, suggesting the critical role of circ_0036627/miR-145/S100A16 axis in PC progression and drug resistance development and offering novel therapeutic targets for PC therapy.

Bayesian inverse problem for a fractional diffusion model of cell migration.

In the present work, both direct and inverse problems are considered for a Fisher-type fractional diffusion equation, which is proposed to describe the phenomenon of cell migration. For the direct problem, a solution is given via the Fourier method and the Laplace transform. On the other hand, we solved the inverse problem from a Bayesian statistical framework using a set of data that are the result of a cell migration experiment on a wound closure assay. We estimated the parameters of the mathematical model via Markov Chain Monte Carlo methods.

ARID1A restrains EMT and stemness of ovarian cancer cells through the Hippo pathway.

Genes encoding subunits of SWI/SNF (BAF) chromatin­remodeling complexes are recurrently mutated in a broad array of tumor types, and among the subunits, ARID1A is the most frequent target with mutations. In the present study, it was reported that ARID1A inhibits the epithelial­mesenchymal transition (EMT) and stemness of ovarian cancer cells, accompanied by reduced cell viability, migration and colony formation, suggesting that ARID1A acts as a tumor suppressor in ovarian cancer. Mechanistically, ARID1A exerts its inhibitory effects on ovarian cancer cells by activating the Hippo signaling pathway. Conversely, the overexpression of a gain­of­function transcriptional co­activator with PDZ­binding motif (TAZ) mutant (TAZ­Ser89) effectively reverses the effects induced by ARID1A. In addition, activation of Hippo signaling apparently upregulates ARID1A protein expression, whereas ectopic expression of TAZ­Ser89 results in the markedly decreased ARID1A levels, indicating a feedback of ARID1A­TAZ in regulating ovarian cancer cell EMT and stemness. Thus, the present study uncovered the role of ARID1A through the Hippo/TAZ pathway in modulating EMT and stemness of ovarian cancer cells, and providing with evidence that TAZ inhibitors could effectively prevent initiation and metastasis of ovarian cancer cases where ARID1A is lost or mutated.

RBM15-Mediated N6-Methyl Adenosine (m6A) Modification of EZH2 Drives the Epithelial-Mesenchymal Transition of Cervical Cancer.

RBM15 functions as an oncogene in multi-type cancers. However, the reports on the roles of RBM15 in cervical cancer are limited. The purpose of this study was to investigate the potentials of RBM15 in cervical cancer. RT-qPCR was conducted to determine mRNA levels. Western was carried out to detect protein expression. CCK-8, colony formation and EdU assays were conducted to determine cell proliferation. Scratch and transwell assays were conducted to determine cell migration and invasion. MeRIP assay was conducted to determine N6-methyl adenosine (m6A) levels. Luciferase assay was conducted to verify the m6A sites of EZH2 and binding sites between EZH2 and promoter of FN1. ChIP assay was conducted to verify the interaction between EZH2 and FN1. The results showed that RBM15 was upregulated in cervical cancer patients and cells. Moreover, high levels of RBM15 predicted poor clinical outcomes. RBM15 knockdown inhibited the proliferation and epithelial-mesenchymal transition (EMT) of cervical cancer cells. RBM15 promoted the m6A modification of EZH2 as well as its protein translation. Additionally, EZH2 bound to the promoter of fibronectin 1 (FN1) and EZH2-FN1 axis is the cascade downstream of RBM15. Overexpressed EZH2 antagonized the effects of RBM15 knockdown and promoted the aggressiveness of cervical cancer cells. In summary, RBM15/EZH2/FN1 signaling cascade induces the proliferation and EMT of cervical cancer. Therefore, RBM15/EZH2/FN1 signaling may be a promising strategy for cervical cancer.

Pan-cancer analysis of DCTN2 and its tumour-promoting role in HCC by modulating the AKT pathway.

Dynactin subunit 2 (DCTN2) has been reported to play a role in progression of several tumours; however, the involvement of DCTN2 in potential mechanism or the tumour immune microenvironment among various cancers still remains largely unknown. Therefore, the objective of this study was to comprehensively investigate the expression status and potential function of DCTN2 in various malignancies through different database, such as The Cancer Genome Atlas, the Genotype-Tissue Expression and Gene Expression Omnimus databases. We discovered that DCTN2 expression was high in many type of tumours tissues compared to adjacent non-tumour ones. High DCTN2 signified poor prognosis for patients with tumours. Additionally, Gene Set Enrichment Analysis (GSEA) analysis revealed that DCTN2 was positively correlated with oncogenic pathways, including cell cycle, tumour metastasis-related pathway, while it was negatively with anti-tumour immune signalling pathway, such as INF-γ response. More importantly, we elucidated the functional impact of DCTN2 on hepatocellular carcinoma (HCC) progression and its underlying mechanisms. DCTN2 expression was much higher in HCC tissues than in adjacent non-tumour tissues. Silencing DCTN2 dramatically suppressed the proliferative and metastasis capacities of tumour cell in vitro. Mechanistically, DCTN2 exerted tumour-promoting effects by modulating the AKT signalling pathway. DCTN2 knockdown in HCC cells inhibited AKT phosphorylation and its downstream targets as well. Rescue experiments revealed that the anti-tumour effects of DCTN2 knockdown were partially reversed upon AKT pathway activation. Overall, DCTN2 may be a potent biomarker signifying tumour prognosis and a promising therapeutic target for tumour treatment, particularly in HCC.

Amentoflavone reverses epithelial-mesenchymal transition in hepatocellular carcinoma cells by targeting p53 signalling pathway axis.

Epithelial-mesenchymal transition (EMT) and its reversal process are important potential mechanisms in the development of HCC. Selaginella doederleinii Hieron is widely used in Traditional Chinese Medicine for the treatment of various tumours and Amentoflavone is its main active ingredient. This study investigates the mechanism of action of Amentoflavone on EMT in hepatocellular carcinoma from the perspective of bioinformatics and network pharmacology. Bioinformatics was used to screen Amentoflavone-regulated EMT genes that are closely related to the prognosis of HCC, and a molecular prediction model was established to assess the prognosis of HCC. The network pharmacology was used to predict the pathway axis regulated by Amentoflavone. Molecular docking of Amentoflavone with corresponding targets was performed. Detection and evaluation of the effects of Amentoflavone on cell proliferation, migration, invasion and apoptosis by CCK-8 kit, wound healing assay, Transwell assay and annexin V-FITC/propidium iodide staining. Eventually three core genes were screened, inculding NR1I2, CDK1 and CHEK1. A total of 590 GO enrichment entries were obtained, and five enrichment results were obtained by KEGG pathway analysis. Genes were mainly enriched in the p53 signalling pathway. The outcomes derived from both the wound healing assay and Transwell assay demonstrated significant inhibition of migration and invasion in HCC cells upon exposure to different concentrations of Amentoflavone. The results of Annexin V-FITC/PI staining assay showed that different concentrations of Amentoflavone induces apoptosis in HCC cells. This study revealed that the mechanism of Amentoflavone reverses EMT in hepatocellular carcinoma, possibly by inhibiting the expression of core genes and blocking the p53 signalling pathway axis to inhibit the migration and invasion of HCC cells.

Microfluidic device reveals new insights into impairment of neutrophil transmigration in patients with sepsis.

Neutrophils need to migrate through tight tissue spaces to eliminate pathogens, but their movement is often hindered by their large and stiff nuclei. Neutrophil migration is impaired in sepsis patients, but it is unclear whether this defect is related to the deformability of their nuclei. Herein, we designed microfluidic devices with micron-scale narrow slits to simulate biological barriers. This setup allowed us to observe and record neutrophil movement and nuclear deformation in real-time. We also developed a method for morphological analysis to quantify nucleus deformation in numerous individual cells. Our studies showed that neutrophils from healthy individuals could adjust their nuclear shape to squeeze through these constrictions, whereas those from sepsis patients demonstrated less flexibility. Neutrophils with rigid nuclei struggled to pass through narrow gaps and were more likely to rupture under pressure. These findings suggest that the migration defects of neutrophils observed in sepsis may be attributed to the inability of neutrophils to deform their nuclei, highlighting the crucial role of microfluidic technologies in offering new insights into migration defects under pathological conditions.

ATF4 inhibits tumor development and mediates p-GCN2/ASNS upregulation in colon cancer.

Colon cancer (CC) is a highly malignant tumor with a high incidence and poor prognosis. This study aimed to explore the function and molecular mechanisms of activating transcription factor 4 (ATF4) in CC. The expression levels of ATF4, GCN2, and ASNS in CC tissues were measured using immunohistochemistry (IHC) and reverse transcription quantitative PCR (RT-qPCR). Cell counting kit-8 (CCK-8), clone formation, transwell, and flow cytometry assays were conducted to assess cell viability, clonogenicity, migration, invasion, cell cycle, and apoptosis, respectively, in the ATF4 knockdown and overexpression SW480 cell lines. The effect of ATF4 on the expression of GCN2 and ASNS was detected using RT-qPCR, Chip-qPCR, and western blotting. ATF4, GCN2, and ASNS were expressed at low levels in CC tissues, and all had a significant negative correlation with tumor diameter. ATF4 knockdown promoted cell proliferation, invasion, and S-phase cell cycle and inhibited apoptosis in SW480 cells. In contrast, ATF4 overexpression had the opposite effect. Furthermore, ATF4 overexpression enhanced ATF4 binding to the ASNS promoter region. ATF4 knockdown significantly inhibited the expression of p-GCN2 and ASNS, whereas ATF4 overexpression significantly upregulated their expression. ATF4 inhibited CC cell viability, clone formation ability, migration, and invasion and promoted apoptosis, possibly by regulating the expression of p-GCN2 and ASNS. Our study provides a novel potential therapeutic target for the treatment of CC.

Role of transforming growth factor-ß1 pathway in angiogenesis induced by chronic stress in colorectal cancer.

BACKGROUND: Chronic stress can induce stress-related hormones; norepinephrine (NE) is considered to have the highest potential in cancer. NE can stimulate the expression of hypoxia-inducible factor-1α (HIF-1α), which is associated with vascular endothelial growth factor (VEGF) secretion and tumor angiogenesis. However, the underlying mechanisms are poorly understood. METHODS: Tumor-bearing mice were subjected to chronic restraint stress and treated with normal saline, human monoclonal VEGF-A neutralizing antibody bevacizumab, or ß-adrenergic receptor (ß-AR) antagonist (propranolol). Tumor growth and vessel density were also evaluated. Human colorectal adenocarcinoma cells were treated with NE, propranolol, or the inhibitor of transforming growth factor-ß (TGF-ß) receptor Type I kinase (Ly2157299) in vitro. TGF-ß1 in mouse serum and cell culture supernatants was quantified using ELISA. The expression of HIF-1α was measured using Real time-PCR and western blotting. Cell migration and invasion were tested. RESULTS: Chronic restraint stress attenuated the efficacy of bevacizumab and promoted tumor growth and angiogenesis in a colorectal tumor model. Propranolol blocked this effect and inhibited TGF-ß1 elevation caused by chronic restraint stress or NE. NE upregulated HIF-1α expression, which was reversed by propranolol or Ly2157299. Propranolol and Ly2157199 blocked NE-stimulated cancer cell migration and invasion. CONCLUSIONS: Our results demonstrate the effect of NE on tumor angiogenesis and the critical role of TGF-ß1 signaling during this process. In addition, ß-AR/TGF-ß1 signaling/HIF-1α/VEGF is a potential signaling pathway. This study also indicates that psychosocial stress might be a risk factor which weakens the efficacy of anti-angiogenic therapy.

MARCH5 promotes hepatocellular carcinoma progression by inducing p53 ubiquitination degradation.

BACKGROUND: Human MARCH5 is a mitochondria-localized E3 ubiquitin-protein ligase that is essential for the regulation of mitochondrial dynamics. A large body of evidence suggests that imbalances in mitochondrial dynamics are strongly associated with cancer. However, the expression, biological function and prognostic significance of MARCH5 in hepatocellular carcinoma (HCC) have not been determined. MATERIALS AND METHODS: The mRNA and protein expression of MARCH5 in HCC cell lines and tumor tissues was assessed by real-time quantitative PCR, Western blot analysis and immunohistochemistry. The clinical prognostic significance of MARCH5 was evaluated in 135 HCC patients. Knockdown or overexpression of MARCH5 in HCC cells was determined by in vitro cell proliferation, migration and invasion assays, and in vivo tumor growth and metastasis assays. In addition, the intrinsic mechanisms by which MARCH5 regulates HCC cell growth and metastasis were explored. RESULTS: MARCH5 was significantly overexpressed in HCC cells and was closely associated with patients' poor postoperative prognosis. In vivo and in vitro experiments revealed that MARCH5 significantly promoted the increase and invasive and migratory ability of hepatocellular carcinoma cells, which was mainly due to the promotion of autophagy by MARCH5. Mechanistic studies revealed that MARCH5 promoted autophagy through ubiquitination degradation of p53 leading to malignant progression of hepatocellular carcinoma. CONCLUSION: Our findings suggest that MARCH5 plays a critical oncogenic role in HCC cells, which provides experimental evidence for the use of MARCH5 as a potential target for HCC therapy.