Cell Scratch Assay Research Articles

Silencing p75NTR regulates osteogenic differentiation and angiogenesis of BMSCs to enhance bone healing in fractured rats

BackgroundFractures heal through a process that involves angiogenesis and osteogenesis but may also lead to non-union or delayed healing. Bone marrow mesenchymal stem cells (BMSCs) have been reported to play a pivotal role in bone formation and vascular regeneration and the p75 neurotrophin receptor (p75NTR) as being an important regulator of osteogenesis. Herein, we aim to determine the potential mediation of BMSCs by p75NTR in bone healing.MethodsRat BMSCs were identified by flow cytometry (FCM) to detect cell cycle and surface markers. Then transfection of si/oe-p75NTR was performed in BMSCs, followed by Alizarin red staining to detect osteogenic differentiation of cells, immunofluorescence double staining was performed to detect the expression of p75NTR and sortilin, co-immunoprecipitation (CO-IP) was conducted to analyze the interaction between p75NTR and sortilin, and EdU staining and cell scratch assay to assess the proliferation and migration of human umbilical vein endothelial cells (HUVECs). The expression of HIF-1α, VEGF, and apoptosis-related proteins were also detected. In addition, a rat fracture healing model was constructed, and BMSCs-si-p75NTR were injected, following which the fracture condition was observed using micro-CT imaging, and the expression of platelet/endothelial cell adhesion molecule-1 (CD31) was assessed.ResultsThe results showed that BMSCs were successfully isolated, p75NTR inhibited apoptosis and the osteogenic differentiation of BMSCs, while si-p75NTR led to a decrease in sortilin expression in BMSCs, increased proliferation and migration in HUVECs, and upregulation of HIF-1α and VEGF expression. In addition, an interaction was observed between p75NTR and sortilin. The knockdown of p75NTR was found to reduce the severity of fracture in rats and increase the expression of CD31 and osteogenesis-related proteins.ConclusionSilencing p75NTR effectively modulates BMSCs to promote osteogenic differentiation and angiogenesis, offering a novel perspective for improving fracture healing.

KLF5 inhibits the migration and invasion in cervical cancer cell lines by regulating SNAI1.

Epithelial-mesenchymal transition (EMT) is an important biological process by which malignant tumor cells to acquire migration and invasion abilities. This study explored the role of KLF5 in the EMT process of in cervical cancer cell lines. Krüpple-like factor 5 (KLF5) is a basic transcriptional factor that plays a key role in cell-cycle arrest and inhibition of apoptosis. However, the molecular mechanism by which KLF5 mediates the biological functions of cervical cancer cell lines has not been elucidated. Here, we focus on the potential function of ELF5 in regulating the EMT process in in vitro model of cervical cancer cell lines. Western-blot and real-time quantitative PCR were used to detect the expression of EMT-related genes in HeLa cells. MTT assays, cell scratch and Transwell assays were used to assess HeLa cells proliferation and invasion capability. Using the bioinformatics tool JASPAR, we identified a high-scoring KLF5-like binding sequence in the SNAI1 gene promoter. Luciferase reporter assays was used to detect transcriptional activity for different SNAI1 promoter truncates. After overexpressing the KLF5 gene in HeLa cells, KLF5 not only significantly inhibited the invasion and migration of HeLa cells, but also increased the expression of E-cadherin and decreased the expression of N-cadherin and MMP9. In addition, the mRNA expression of upstream regulators of E-cadherin, such as SNAI1, SLUG, ZEB1/2 and TWIST1 was also decreased. Furthermore, KLF5 inhibiting the expression of the SNAI1 gene via binding its promoter region, and the EMT of Hela cells was promoted after overexpression of the SNAI1 gene. These results indicate that KLF5 can downregulate the EMT process of HeLa cells by decreasing the expression of the SNAI1 gene, thereby inhibiting the migration and invasion of HeLa cervical cancer cells.

LncRNA SNHG11 promotes malignant progression of colorectal cancer cells through the PI3K/Akt/mTOR signaling pathway

Objective: To investigate the effects of lncRNA SNHG11 on proliferation, migration, invasion and apoptosis of colorectal cancer cancer cells and possible mechanisms. Methods: qRT-PCR was performed to detect the expression level of lncRNA SNHG11 in colorectal cancer tissues and its related cell lines. The correlation between SNHG11 expression and clinical prognosis of patients was assessed by bioinformatics techniques. Cultured CRC cell lines were transfected with shCtrl (shCtrl group), shSNHG11#1 (shSNHG11#1 group), shSNHG11#2 (shSNHG11#2 group), Control cDNA (Control cDNA group), and SNHG11 cDNA (SNHG11 cDNA), respectively. Thiazolyl blue (MTT), clone formation assay, Transwell assay, cell scratch assay, and flow cytometry were used to detect the proliferation, migration, invasion, and apoptosis of CRC cells in each group. Western protein blotting was used to detect the expression of relevant proteins in each group, and the effect of lncRNA SNHG11 knockdown on the growth of tumour cells in vivo was analysed by nude mice tumouring assay. Phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signalling pathway inhibitor LY294002 was used for rescue experiments. Results: The expression of lncRNA SNHG11 was significantly higher in colorectal cancer cells and tissues than in normal tissues (P<0.05). Survival analysis showed that the expression level of SNHG11 was not statistically associated with CRC survival (P>0.05). shSNHG11#2 group compared with shCtrl group. MTT OD490/570 values decreased, the number of CRC cell clones decreased, the number of Transwell cells decreased, the area of cell scratch decreased, and the apoptosis rate increased (P<0.05). The mesenchymal markers matrix metalloproteinase (MMP9), N-cadherin and vimentin were significantly reduced, and the expression of the epithelial marker E-cadherin was upregulated. The expression of anti-apoptotic proteins Bcl-2 and Bcl-xl was decreased, and the expression of pro-apoptotic protein Bax was increased (P<0.05).In vivo experiments showed that lncRNA SNHG11 knockdown inhibited the growth of colorectal cancer cells, and the expression of Ki67 was reduced in tumours (P<0.05). LncRNA SNHG11 knockdown inhibited the expression of p-PI3K, p-Akt and p-mTOR.The PI3K/Akt/mTOR signaling pathway inhibitor LY294002 was able to restore the malignant cytological progression of colorectal cancer cells induced by the overexpression of lncRNA SNHG11. Conclusions: LncRNA SNHG11 is highly expressed in colorectal cancer. lncRNA SNHG11 can promote the malignant progression of colorectal cancer cells by regulating the PI3K/Akt/mTOR signaling pathway, and this finding provides a new theoretical basis for targeted therapy of colorectal cancer.

Nobiletin alleviates atherosclerosis by inhibiting lipid uptake via the PPARG/CD36 pathway

BackgroundAtherosclerosis (AS) is a persistent inflammatory condition triggered and exacerbated by several factors including lipid accumulation, endothelial dysfunction and macrophages infiltration. Nobiletin (NOB) has been reported to alleviate atherosclerosis; however, the underlying mechanism remains incompletely understood.MethodsThis study involved comprehensive bioinformatic analysis, including multidatabase target prediction; GO and KEGG enrichment analyses for function and pathway exploration; DeepSite and AutoDock for drug binding site prediction; and CIBERSORT for immune cell involvement. In addition, target intervention was verified via cell scratch assays, oil red O staining, ELISA, flow cytometry, qRT‒PCR and Western blotting. In addition, by establishing a mouse model of AS, it was demonstrated that NOB attenuated lipid accumulation and the extent of atherosclerotic lesions.Results(1) Altogether, 141 potentially targetable genes were identified through which NOB could intervene in atherosclerosis. (2) Lipid and atherosclerosis, fluid shear stress and atherosclerosis may be the dominant pathways and potential mechanisms. (3) ALB, AKT1, CASP3 and 7 other genes were identified as the top 10 target genes. (4) Six genes, including PPARG, MMP9, SRC and 3 other genes, were related to the M0 fraction. (5) CD36 and PPARG were upregulated in atherosclerosis samples compared to the normal control. (6) By inhibiting lipid uptake in RAW264.7 cells, NOB prevents the formation of foam cell. (7) In RAW264.7 cells, the inhibitory effect of oxidized low-density lipoprotein on foam cells formation and lipid accumulation was closely associated with the PPARG signaling pathway. (8) In vivo validation showed that NOB significantly attenuated intra-arterial lipid accumulation and macrophage infiltration and reduced CD36 expression.ConclusionsNobiletin alleviates atherosclerosis by inhibiting lipid uptake via the PPARG/CD36 pathway.

ZNF692 drives malignant development of hepatocellular carcinoma cells by promoting ALDOA-dependent glycolysis

Hepatocellular carcinoma (HCC) is one of the malignancies with the worst prognosis worldwide, in the occurrence and development of which glycolysis plays a central role. This study uncovered a mechanism by which ZNF692 regulates ALDOA-dependent glycolysis in HCC cells. RT-qPCR and western blotting were used to detect the expression of ZNF692, KAT5, and ALDOA in HCC cell lines and a normal liver cell line. The influences of transfection-induced alterations in the expression of ZNF692, KAT5, and ALDOA on the functions of HepG2 cells were detected by performing MTT, flow cytometry, Transwell, cell scratch, and colony formation assays, and the levels of glucose and lactate were determined using assay kits. ChIP and luciferase reporter assays were conducted to validate the binding of ZNF692 to the KAT5 promoter, and co-IP assays to detect the interaction between KAT5 and ALDOA and the acetylation of ALDOA. ZNF692, KAT5, and ALDOA were highly expressed in human HCC samples and cell lines, and their expression levels were positively correlated in HCC. ZNF692, ALDOA, or KAT5 knockdown inhibited glycolysis, proliferation, invasion, and migration and promoted apoptosis in HepG2 cells. ZNF692 bound to the KAT5 promoter and promoted its activity. ALDOA acetylation levels were elevated in HCC cell lines. KAT5 bound to ALDOA and catalyzed ALDOA acetylation. ALDOA or KAT5 overexpression in the same time of ZNF692 knockdown, compared to ZNF692 knockdown only, stimulated glycolysis, proliferation, invasion, and migration and reduced apoptosis in HepG2 cells. ZNF692 promotes the acetylation modification and protein expression of ALDOA by catalyzing KAT5 transcription, thereby accelerating glycolysis to drive HCC cell development.

Circ_0082374 Promotes the Tumorigenesis and Suppresses Ferroptosis in Non-small Cell Lung Cancer by Up-Regulating GPX4 Through Sequestering miR-491-5p.

Circular RNAs (circRNAs) have been identified to be dysregulated in non-small cell lung cancer (NSCLC) and implicated in the progression of this cancer. Here, this work aimed to investigate the role and mechanism of circ_0082374 on NSCLC progression. Levels of circ_0082374, miR-491-5p, GPX4 (glutathione peroxidase 4) and epithelial-mesenchymal transition (EMT)-related proteins were examined by quantitative real-time PCR or western blotting, respectively. Cell proliferation and metastasis were detected using cell counting kit-8, colony formation, EdU, transwell, and Scratch assays. Cell ferroptosis was evaluated by measuring cell survival after the treatment of different ferroptosis inducers or inhibitors, as well as the accumulation of intracellular reactive oxygen species (ROS), ferrous iron (Fe2+) and malondialdehyde (MDA). The binding between miR-491-5p and circ_0082374 or GPX4 was confirmed using dual-luciferase reporter and RNA pull-down assays. In vivo experiments were conducted using murine xenograft assay and immunohistochemistry. Circ_0082374 was a stable circRNA with high expression in NSCLC tissues and cells. Functionally, circ_0082374 silencing suppressed NSCLC cell proliferation and metastasis. Moreover, its down-regulation enhanced ferroptosis by decreasing iron and lipid peroxidation accumulation. Mechanistically, circ_0082374 could indirectly up-regulate GPX4 expression via miR-491-5p, indicating the circ_0082374/miR-491-5p/GPX4 competitive endogenous RNAs (ceRNA) network. Rescue experiments demonstrated that the miR-491-5p/GPX4 axis mediated the regulatory effects of circ_0082374 exerted on NSCLC cells. Moreover, knockdown of circ_0082374 impeded NSCLC growth and EMT via regulating miR-491-5p and GPX4. Circ_0082374 silencing could suppress NSCLC cell proliferation, metastasis and induce ferroptosis through miR-491-5p/GPX4 axis, suggesting a novel therapeutic approach for NSCLC patients.

Long noncoding RNA GPRC5D-AS1 in renal cell carcinoma: a molecular mechanism study.

Clear cell renal cell carcinoma (RCC) is the most common subtype of RCC. Although targeted therapy can provide superior treatment outcomes, it is prone to drug resistance, and individual responses to immunotherapy vary greatly. Therefore, finding new diagnostic and therapeutic targets for RCC is of considerable importance. Long noncoding RNA (lncRNA) GPRC5D-AS1 can serve as a biomarker in clinical applications and the prognosis of lung squamous cell carcinoma. However, the specific mechanism of action of lncRNA GPRC5D-AS1 in RCC has not yet been clarified. Therefore, this paper explores the expression of lncRNA GPRC5D-AS1 in the renal cancer cell line 786-0, and conducts a preliminary study of its molecular mechanism. Selecting nude mice for tumor experiments is because of the high genomic and physiological similarity between mice and humans. Conducting tumor research on mice allows for better control of experimental conditions, aiding researchers in more accurately observing and analysing tumor characteristics and responses. Small interfering RNA (siRNA) and plasmid cloning DNA (pcDNA) 3.1 were used to transfect renal cancer cell line 786-0 to silence and overexpress the lncRNA GPRC5D-AS1 gene. Quantitative real-time fluorescence polymerase chain reaction was used to detect the difference in lncRNA GPRC5D-AS1 expression in blank control group, negative control group, siGPRC5D-AS1 group and oeGPRC5D-AS1 group. The effects of silence and overexpression of lncRNA GPRC5D-AS11 on the proliferation of 786-0 cells were detected in cell colony formation experiments; the changes in the migration and invasion of 786-0 cells were detected via cell scratch assay and transwell assay, respectively; the differences in tumor growth between groups were determined via tumorigenesis experiments in nude mice; and the expression of proliferation-related protein [β-catenin, Ki67 and proliferating cell nuclear antigen (PCNA)] and invasion-related protein (N-cadherin and E-cadherin) were detected via Western blotting. Compared with blank control group and negative control group, the siGPRC5D-AS1 group showed a significant decrease in the relative expression of lncRNA GPRC5D-AS1 (P<0.05), a significant increase in the number of proliferating cells and migrating cells (P<0.05), a significant increase in the tumor volume of nude mice (P<0.05), a significant increase in β-catenin, Ki67, PCNA and N-cadherin protein expression (P<0.05), and a significant decrease in E-cadherin protein expression (P<0.05); conversely, these results were opposite for the eGPRC5D-AS1 group. Silencing the expression of lncRNA GPRC5D-AS1 can enhance the proliferation, invasion, and migration ability of renal cancer cell line 786-0, which can be weakened by the overexpression of lncRNA GPRC5D-AS1.

Preparation and Evaluation of Folate Modified PEG-PLLA Nanoparticles Loaded with Lycorine for Glioma Treatment.

Lycorine is a kind of natural active ingredient with a strong antitumor effect. In this study, folate ligand-conjugated polyethylene glycol-block-poly(l-lactide) (PEG-PLLA) nanoparticles (FA-PEG-PLLA NPs) were designed to deliver lycorine to enhance its anti-glioma activity. The successful preparation of the FA-PEG-PLLA polymer was confirmed by 1H-NMR, FT-IR, XRD, TGA, and DSC. The optimal formulation for LYC@FA-PEG-PLLA NPs was determined by response surface analysis as follows: sodium dodecyl sulfate (SDS) of 1%, carrier material of 0.03 g, organic phase volume of 3 mL, and ultrasonic power of 20%. The LYC@FA-PEG-PLLA NPs exhibited an encapsulation efficiency of 83.58% and a particle size of 49.71 nm, demonstrating good stability. Hemolysis experiments, MTT assays, and cell scratch assays revealed excellent biocompatibility of FA-PEG-PLLA and superior anti-glioma activity of LYC@FA-PEG-PLLA NPs compared to the raw drug. Additionally, cell apoptosis assays, ROS experiments, and western blot analysis demonstrated that LYC@FA-PEG-PLLA NPs contributed to cell apoptosis by inducing ROS generation and increasing the expression of NF-κB inhibitory protein IκBα. These results suggested that LYC@FA-PEG-PLLA NPs hold promise for glioma treatment.

HOTAIR/miR-1277-5p/FBN2 signaling axis is involved in recurrent spontaneous abortion by regulating the growth, migration, and invasion of HTR-8/SVneo cells†.

This study aimed to explore the specific pathways by which HOX transcript antisense intergenic RNA contributes to the pathogenesis of unexplained recurrent spontaneous abortion. Real-time quantitative PCR was employed to assess the differential expression levels of HOX transcript antisense intergenic RNA in chorionic villi tissues from unexplained recurrent spontaneous abortion patients and women with voluntarily terminated pregnancies. HTR-8/SVneo served as a cellular model. Knockdown and overexpression of HOX transcript antisense intergenic RNA in the cells were achieved through siRNA transfection and pcDNA3.1 transfection, respectively. Cell viability, migration, and invasion were evaluated using cell counting kit-8, scratch, and Transwell assays, respectively. The interaction among the HOX transcript antisense intergenic RNA /miR-1277-5p/fibrillin 2 axis was predicted through bioinformatics analysis and confirmed through in vitro experiments. Furthermore, the regulatory effects of the HOX transcript antisense intergenic RNA /miR-1277-5p/fibrillin 2 signaling axis on cellular behaviors were validated in HTR-8/SVneo cells. We found that HOX transcript antisense intergenic RNA was downregulated in chorionic villi tissues from unexplained recurrent spontaneous abortion patients. Overexpression of HOX transcript antisense intergenic RNA significantly enhanced the viability, migration, and invasion of HTR-8/SVneo cells, while knockdown of HOX transcript antisense intergenic RNA had the opposite effects. We further confirmed the regulatory effect of the HOX transcript antisense intergenic RNA /miR-1277-5p/fibrillin 2 signaling axis in unexplained recurrent spontaneous abortion. Specifically, HOX transcript antisense intergenic RNA and fibrillin 2 were found to reduce the risk of unexplained recurrent spontaneous abortion by enhancing cell viability, migration, and invasion, whereas miR-1277-5p exerted the opposite effects. HOX transcript antisense intergenic RNA promotes unexplained recurrent spontaneous abortion development by targeting inhibition of miR-1277-5p/fibrillin 2 axis.

Demethylzeylasteral inhibits proliferation, migration and invasion and promotes apoptosis of non-small cell lung cancer cells by inhibiting the AKT/CREB signaling pathway

To investigate the mechanism underlying the inhibitory effects of Demethylzeylasteral (T-96) on non-small cell lung cancer (NSCLC) cells. We first examined the effects of different concentrations (1, 3, 10, and 30 μmol/L) of demethylzeylasteral on morphology and cell number of A549 and H1299 cells. The changes in proliferation, cell viability, migration, invasion, and apoptosis of A549 and H1299 cells following demethylzeylasteral treatment were detected using clone formation, CCK-8, cell scratch, Transwell, and flow cytometric assays, and the effect of SC79 treatment against demethylzeylasteral-induced cell apoptosis was assessed. Western blotting was performed to detect the changes in expressions of E-cadherin, N-cadherin, vimentin, Bax, Bcl-2 and cleaved caspase-3 and phosphorylation of AKT/CREB in demethylzeylasteral-treated A549 and H1299 cells and the cellular expressions of apoptotic proteins following treatment with both demethylzeylasteral and SC79. T-96 treatment caused elongation of the cell body and widening of the intercellular space and significantly inhibited cell viability, proliferation, migration and invasion of A549 and H1299 cells (P < 0.05). Flow cytometry showed that demethylzeylasteral induced apoptosis in both A549 and H1299 cells, whereas SC79 treatment obviously attenuated its pro-apoptotic effect (P < 0.05). Western blotting revealed up-regulated expressions of Bax and cleaved caspase-3 proteins and lowered Bcl-2 expression level in demethylzeylasteral-treated A549 and H1299 cells, but cotreatment with SC79 obviously attenuated the expressions of the apoptotic proteins. T-96 significantly up-regulated the expression level of E-cadherin, down-regulated the expressions of N-cadherin and vimentin, and inhibited the phosphorylation of AKT and CREB in the two cell lines (P < 0.05). T-96 inhibits the proliferation, migration and invasion and induces apoptosis of NSCLC cells possibly by inhibiting the AKT/CREB signaling pathway.

Immune-related long noncoding RNA zinc finger protein 710-AS1-201 promotes the metastasis and invasion of gastric cancer cells.

Gastric cancer (GC) is a prevalent malignant tumor of the gastrointestinal system. ZNF710 is a transcription factor (TF), and zinc finger protein 710 (ZNF710)-AS1-201 is an immune-related long noncoding RNA (lncRNA) that is upregulated in GC cells. To assess the correlation between ZNF710-AS1-201 and immune microenvironment features and to investigate the roles of ZNF710-AS1-201 in the invasion and metastasis processes of GC cells. We obtained data from The Cancer Genome Atlas and Wujin Hospital. We assessed cell growth, migration, invasion, and programmed cell death using cell counting kit-8, EdU, scratch, Transwell, and flow cytometry assays. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to identify the potential downstream targets of ZNF710-AS1-201. In GC tissues with low ZNF710-AS1-201 expression, immunoassays detected significant infiltration of various antitumor immune cells, such as memory CD8 T cells and activated CD4 T cells. In the low-expression group, the half-maximal inhibitory concentrations (IC50s) of 5-fluorouracil, cisplatin, gemcitabine, and trametinib were lower, whereas the IC50s of dasatinib and vorinostat were higher. The malignant degree of GC was higher and the stage was later in the high-expression group. Additionally, patients with high expression of ZNF710-AS1-201 had lower overall survival and disease-free survival rates. In vitro, the overexpression of ZNF710-AS1-201 greatly enhanced growth, metastasis, and infiltration while suppressing cell death in HGC-27 cells. In contrast, the reduced expression of ZNF710-AS1-201 greatly hindered cell growth, enhanced apoptosis, and suppressed the metastasis and invasion of MKN-45 cells. The expression changes in ZNF710 were significant, but the corresponding changes in isocitrate dehydrogenase-2, Semaphorin 4B, ARHGAP10, RGMB, hsa-miR-93-5p, and ZNF710-AS1-202 were not consistent or statistically significant after overexpression or knockdown of ZNF710-AS1-201, as determined by qRT-PCR. Immune-related lncRNA ZNF710-AS1-201 facilitates the metastasis and invasion of GC cells. It appears that ZNF710-AS1-201 and ZNF710 have potential as effective targets for therapeutic intervention in GC. Nevertheless, it is still necessary to determine the specific targets of the ZNF710 TF.

Reduced Proliferative Capacity and Defense against Staphylococcus aureus in Human Nasal Mucosal Epithelium Lacking ZNF365

Introduction: Chronic rhinosinusitis with nasal polyps (CRSwNP) is a common chronic inflammatory disease of the nose characterized by barrier disruption and environmental susceptibility, and the deletion of ZNF365 may be a factor inducing these manifestations. However, there is no study on the mechanism of action between CRSwNP and ZNF365. Therefore, this study focuses on the effect of the zinc finger protein ZNF365 on the proliferation of nasal mucosal epithelial cells and their defense against Staphylococcus aureus (S. aureus). Methods: Immunohistochemistry and Western blot were applied to verify the changes of ZNF365 expression in nasal polyp tissues and control tissues, as well as in primary epithelial cells. ZNF365 was knocked down in human nasal mucosa epithelial cell line (HNEpc), and the proliferation, migration, and transdifferentiation of epithelium were observed by immunofluorescence, QPCR, CCK8, and cell scratch assay. The changes of mesenchymal markers and TLR4-MAPK-NF-κB pathway were also observed after the addition of S. aureus. Results: ZNF365 expression was reduced in NP tissues and primary nasal mucosal epithelial cells compared to controls. Knockdown of ZNF365 in HNEpc resulted in decreased proliferation and migration ability of epithelial cells and abnormal epithelial differentiation (decreased expression of tight junction proteins). S. aureus stimulation further inhibited epithelial cell proliferation and migration, while elevated markers of epithelial-mesenchymal transition and inflammatory responses occurred. Conclusion: ZNF365 is instrumental in maintaining the proliferative capacity of nasal mucosal epithelial cells and defending against the invasion of S. aureus. The findings suggest that ZNF365 may participate in the development of CRSwNP.

Overexpression of miR-506-3p reversed doxorubicin resistance in drug-resistant osteosarcoma cells.

Background and objective: Osteosarcoma is a common primary malignant tumor of bone, and doxorubicin is one of the most widely used therapeutic drugs. While the problem of doxorubicin resistance limits the long-term treatment benefits in osteosarcoma patients. The role of miRNAs and their target genes in osteosarcoma have become increasingly prominent. Currently, there is no report on miR-506-3p reversing doxorubicin resistance by targeting STAT3 in osteosarcoma. The purpose of this study was to investigate the molecular mechanism that overexpression of miR-506-3p reverses doxorubicin resistance in drug-resistant osteosarcoma cells. Methods: Doxorubicin-resistant osteosarcoma cells (U-2OS/Dox) were constructed by intermittent stepwise increasing stoichiometry. The target genes of miR-506-3p were predicted by bioinformatics approach and the targeting relationship between miR-506-3p and STAT3 was detected using dual luciferase reporter assay. U-2OS/Dox cells were treated with miR-506-3p overexpression and STAT3 silencing respectively. Then Western blot and RT-qPCR were used to detect the protein and mRNA expression levels of JAK2/STAT3 signaling pathway, drug-resistant and apoptotic associated molecules. The migration and invasion were assessed by cell scratch assay and transwell assay. The cell proliferative viability and apoptosis were investigated by CCK8 assay and flow cytometry assay. Results: U-2OS/Dox cells were successfully constructed with a 14.4-fold resistance. MiR-506-3p is directly bound to the 3'-UTR of STAT3 mRNA. Compared with U-2OS cells, the mRNA expression of miR-506-3p was reduced in U-2OS/Dox cells. Overexpression of miR-506-3p decreased the mRNA expression levels of JAK2, STAT3, MDR1/ABCB1, MRP1/ABCC1, Survivin and Bcl-2, and decreased the protein expression levels of p-JAK2, STAT3, MDR1/ABCB1, MRP1/ABCC1, Survivin and Bcl-2, and conversely increased Bax expression. It also inhibited the proliferation, migration and invasion of U-2OS/Dox cells and promoted cells apoptosis. The results of STAT3 silencing experiments in the above indicators were consistent with that of miR-506-3p overexpression. Conclusion: Overexpression of miR-506-3p could inhibit the JAK2/STAT3 pathway and the malignant biological behaviors, then further reverse doxorubicin resistance in drug-resistant osteosarcoma cells. The study reported a new molecular mechanism for reversing the resistance of osteosarcoma to doxorubicin chemotherapy and provided theoretical support for solving the clinical problems of doxorubicin resistance in osteosarcoma.

Circulating microvesicles miR139-3p from bronchopulmonary dysplasia aggravates pulmonary vascular simplification by targeting 4E binding protein 1.

Microvesicles (MVs) play a crucial role in bronchopulmonary dysplasia (BPD). There are many MVs in circulating plasma, and they are in direct contact with lung endothelial cells. However, the molecular mechanism and causative effect of circulating MVs on BPD remain unclear. Clinical plasma samples were collected, circulating MVs were isolated, and microRNA (miRNA) sequencing was performed. The BPD model was established, and different MVs were administered. Alveoli and pulmonary vessels were examined by hematoxylin-eosin staining, and body weight and length were measured. In vitro, gene expression was disrupted by miRNA mimics, miRNA inhibitors or plasmid transfection. Cell proliferation and protein expression were detected by cell scratch assay, accurate 5-ethynyl-2-deoxyuridine test, western blotting, or immunofluorescence assay. BPD-derived MVs further aggravated pulmonary vascular simplification, while circulating MVs from control mice mitigated pulmonary vascular simplification. Micro-RNA sequencing and independent sample verification revealed that miR139-3p, but not miR6125 or miR193b-3p, was the most critical effector molecule in MVs. Mechanism studies showed that eukaryotic translation initiation factor 4E binding protein 1 was the target gene for miR139-3p. In addition, we found that supplementation of miR139-3p inhibitor partially alleviated pulmonary vascular simplification. These results indicate that circulating MVs are involved in forming BPD by carrying miR139-3p molecules and support miR139-3p inhibitors as a potential therapeutic strategy for alleviating pulmonary vascular simplification in BPD.

Redox-responsive Nanomicelles with Intracellular Targeting and Programmable Drug Release for Targeted Tumor Therapy.

Anti-inflammatory medications, in particular aspirin, have chemopreventive and anticancer adjuvant effects on specific types of cancers, according to ongoing anti-tumor research. Additionally, efforts have been made to transform Poly(salicylic acid) (PSA) into delivery-related nanocarriers. to transport anticancer medications into nanocarriers. However, tumor cell targeting and tumor selectivity were lacking in the salicylic acid polymer-based nanocarriers, preventing them from performing to their full potential. The objective of this study is to prepare targeting and reduction-responsive poly pre-drug nanocarriers (HA-ss-PSA NPs) and to investigate the feasibility of delivering adriamycin (DOX) as nanocarriers. The structures of the polymers were confirmed by nuclear magnetic resonance hydrogen spectroscopy (1H-NMR) and infrared spectroscopy (IR); the encapsulation rate and drug loading of DOX-loaded nanoparticles were determined by HPLC; and the anti-tumor effects of the carriers were evaluated by MTT experiments and in vivo</i> experiments. The prepared nanocarriers had uniform particle size distribution. The drug release rate was up to 80% within 48 h in the tumor environment. DOX/HA-ss-PSA NPs showed significant cytostatic effects. In addition, HA-ss-PSA NPs showed significant targeting and inhibition of cell migration in cell uptake and scratch assays. In vivo</i> experiments showed that the prepared carriers had high tumor inhibition rates, good targeting effects on the liver and tumor, and significantly reduced toxicity to other tissues. The prepared HA-ss-PSA NPs could effectively inhibit the growth of HepG2 cells and tumors in vivo</i>, indicating that PSA could be used as a backbone component of a safe and reliable drug delivery system, providing a new strategy for the treatment of liver cancer.

Mechanism of astragaloside Ⅳ combined with Panax notoginseng saponins in regulating angiogenesis to treat cerebral ischemia based on network pharmacology and experimental verification

Network pharmacology and animal and cell experiments were employed to explore the mechanism of astragaloside Ⅳ(AST Ⅳ) combined with Panax notoginseng saponins(PNS) in regulating angiogenesis to treat cerebral ischemia. The method of network pharmacology was used to predict the possible mechanisms of AST Ⅳ and PNS in treating cerebral ischemia by mediating angiogenesis. In vivo experiment: SD rats were randomized into sham, model, and AST Ⅳ(10 mg·kg~(-1)) + PNS(25 mg·kg~(-1)) groups, and the model of cerebral ischemia was established with middle cerebral artery occlusion(MCAO) method. AST Ⅳ and PNS were administered by gavage twice a day. the Longa method was employed to measure the neurological deficits. The brain tissue was stained with hematoxylin-eosin(HE) to reveal the pathological damage. Immunohistochemical assay was employed to measure the expression of von Willebrand factor(vWF), and immunofluorescence assay to measure the expression of vascular endothelial growth factor A(VEGFA). Western blot was employed to determine the protein levels of vascular endothelial growth factor receptor 2(VEGFR2), VEGFA, phosphorylated phosphatidylinositol 3-kinase(p-PI3K), and phosphorylated protein kinase B(p-AKT) in the brain tissue. In vitro experiment: the primary generation of rat brain microvascular endothelial cells(rBEMCs) was cultured and identified. The third-generation rBMECs were assigned into control, model, AST Ⅳ(50 μmol·L~(-1)) + PNS(30 μmol·L~(-1)), LY294002(PI3K/AKT signaling pathway inhibitor), 740Y-P(PI3K/AKT signaling pathway agonist), AST Ⅳ + PNS + LY294002, and AST Ⅳ + PNS + 740Y-P groups. Oxygen glucose deprivation/re-oxygenation(OGD/R) was employed to establish the cell model of cerebral ischemia-reperfusion injury. The cell counting kit-8(CCK-8) and scratch assay were employed to examine the survival and migration of rBEMCs, respectively. Matrigel was used to evaluate the tube formation from rBEMCs. The Transwell assay was employed to examine endothelial cell permeability. Western blot was employed to determine the expression of VEGFR2, VEGFA, p-PI3K, and p-AKT in rBEMCs. The results of network pharmacology analysis showed that AST Ⅳ and PNS regulated 21 targets including VEGFA and AKT1 of angiogenesis in cerebral infarction. Most of these 21 targets were involved in the PI3K/AKT signaling pathway. The in vivo experiments showed that compared with the model group, AST Ⅳ + PNS reduced the neurological deficit score(P&lt;0.05) and the cell damage rate in the brain tissue(P&lt;0.05), promoted the expression of vWF and VEGFA(P&lt;0.01) and angiogenesis, and up-regulated the expression of proteins in the PI3K/AKT pathway(P&lt;0.05, P&lt;0.01). The in vitro experiments showed that compared with the model group, the AST Ⅳ + PNS, 740Y-P, AST Ⅳ + PNS + LY294002, and AST Ⅳ + PNS + 740Y-P improved the survival of rBEMCs after OGD/R, enhanced the migration of rBEMCs, increased the tubes formed by rBEMCs, up-regulated the expression of proteins in the PI3K/AKT pathway, and reduced endothelial cell permeability(P&lt;0.05, P&lt;0.01). Compared with the LY294002 group, the AST Ⅳ + PNS + LY294002 group showed increased survival rate, migration rate, and number of tubes, up-regulated expression of proteins in the PI3K/AKT pathway, and decreased endothelial cell permeability(P&lt;0.05,P&lt;0.01). Compared with the AST Ⅳ + PNS and 740Y-P groups, the AST Ⅳ + PNS + 740Y-P group presented increased survival rate, migration rate, and number of tubes and up-regulated expression of proteins in the PI3K/AKT pathway, and reduced endothelial cell permeability(P&lt;0.01). This study indicates that AST Ⅳ and PNS can promote angiogenesis after cerebral ischemia by activating the PI3K/AKT signaling pathway.

Melt electrowriting (MEW)-PCL composite Three-Dimensional exosome hydrogel scaffold for wound healing

While mesenchymal stem cell-derived exosomes hold substantial potential in wound healing, challenges persist in terms of large-scale production and activity of 2D-culture derived exosome, as well as addressing their inactivation and loss during application. 3D exosomes can be produced more efficiently and possess higher activity. However, there lacks a delivery patch mimicking nanofibrous architecture of the extracellular matrix while facilitating the in situ delivery of exosomes, thereby minimizing dissipation of exosomes and accelerating the process of wound healing. In this study, we devised a controllable GelMA hydrogel-combined Melt Electrowriting (MEW)-PCL scaffold for in situ 3D-exosome release. We showed that biocompatible scaffolds prepared by MEW have a simulated extracellular matrix with a highly controllable arrangement of nanofibers that can support cell adhesion, proliferation and differentiation. Through cell proliferation, scratch assay, and tube formation experiments, we verified that 3D exosomes could effectively stimulate cell proliferation, migration, and tube formation, with dose-dependent effects. In vivo outcomes exhibited accelerated re-epithelialization, improved collagen maturation, and enhanced angiogenesis. Our findings suggest that 3D-cultured exosomes within the scaffold significantly enhance wound repair. This innovative delivery strategy opens up new avenues for the application of MSC-derived exosomes in wound healing.

Co-treatment of silymarin and cisplatin inhibited cell proliferation, induced apoptosis in ovarian cancer.

Ovarian cancer is one of the most lethal gynecological cancers among women worldwide. Cisplatin (Cis) is an effective chemotherapeutic agent used to treat several types of cancer. Silymarin (SLM) is an extract of medicinal plant Silybum marianum (milk thistle) with anti-inflammatory, anti-angiogenesis, antioxidant, and anticancer properties used alone or in combination with other drugs. This study aimed to explore the effects of co-treatment with SLM and Cis on A2780 human ovarian cancer cell lines. In this study, A2780 cells were treated with various concentrations of SLM and Cis, separately and in combination. Cell cytotoxicity, scratch, clonogenic, and flow-cytometry assays were accomplished to estimate cell viability, migration, colony formation, and apoptosis, respectively. Real-time PCR was utilized to determine the expression levels of miR-155 and miR-27a. SLM significantly reduced the proliferation of A2780 cells in a concentration- and time-dependent manner. Combination treatment with SLM and Cis was more potent than either single treatment in reducing viability, suppressing migration, inhibiting colony formation, and promoting the induction of apoptosis. Additionally, gene expression analysis revealed a significant decline in the expression levels of miR-155 and miR-27a in response to all separate and combined treatments, and co-treatment was more effective than individual treatments in altering miRNAs expression. Based on our findings, SLM boosts the anticancer activity of Cis and mitigates its side effects. Thus, the co-treatment of SLM and Cis can be proposed as a promising therapeutic strategy for further investigation.

Arı zehiri bileşenlerinin akciğer kanserine karşı antikanser etkisi

This study aims to determine the effects of bee venom on the proliferation capacity of Calu-3 cells and the migration ability of the cells. For this purpose, bee venom samples were collected from Apis mellifera anatoliaca in Muğla (Türkiye) provinces and Calu-3 cells were exposed to this bee venom. A test for cell viability using Calu-3 given bee venom in varied doses (20 μg/mL, 18 μg/mL, 15 μg/mL, 12 μg/mL, 10 μg/mL, 9 μg/mL, 7.5 μg/mL, 5 μg/mL, 2.5 μg/mL, 1.25 μg/mL, 0.625 μg/mL and 0.312 μg/mL) was conducted. And scratch assay was performed on cells treated with the doses (15 μg/mL, 10 μg/mL, 7.5 μg/mL, 1.25 and 0.312 μg/mL) and imaged every two hours for 24 hours. According to the results of our study's cell proliferation and scratch assays, bee venom had a cytotoxic and proliferative effect on Calu-3 cells which had a dose-dependent cytotoxic and proliferative effect. The study's outcomes how crucial dosage adjustment is in the use of bee venom in lung cancer studies due to its cytotoxic effect. Even though we have achieved a better understanding of how bee venom components work, our knowledge might still be improved by looking at how bee venom affects Calu-3 cells when combined with other substances or by developing the purification method for bee venom.

Ropivacaine combined with sorafenib attenuates hepatocellular carcinoma cell proliferation and metastasis by inhibiting the miR-224/HOXD10 axis.

The development of drug resistance in hepatocellular carcinoma (HCC) cells limits the effectiveness of sorafenib (Sor). However, the regulatory mechanisms underlying the effects of the combination Sor and ropivacaine (Rop) on HCC cells remain unclear. miR-224 and HOXD10 mRNA expression in HCC cells was analyzed using qRT-PCR. CCK-8, Transwell assays and tumor formation experiments in nude mice were used to assess HCC cell proliferation, migration, and invasion. Migration of HCC cells was also analyzed using a cell scratch assay. Hematoxylin and eosin staining was used to detect tumor area. miR-224 expression profoundly increased in HepG2 and Huh7 cells. Treatment with Rop and/or Sor blocked miR-244 expression, especially the combination treatment. Transfection of miR-224 mimic increased HCC cell proliferation and tumor size in nude mice, and migration and invasion in vitro in the presence of Rop and Sor compared to the negative control mimic. Dual-luciferase reporter assays showed that HOXD10 was targeted by miR-224. HOXD10 protein expression and was markedly reduced in HepG2 and Huh7 cells. Rop and/or Sor treatment increased HOXD10 protein expression, particularly the combination treatment. miR-224 negatively regulated HOXD10 expression in HCC cells treated with Rop and Sor. Transfection-mediated silencing of HOXD10 increased HCC cell proliferation, migration, and invasion in the presence of Rop and Sor compared with negative control transfection. The combination of Rop and Sor attenuates HCC cell proliferation and metastasis via the miR-224/HOXD10 axis.