Cell Counting Kit-8 Test Research Articles

Effects of BRCA1 overexpression via the NRF2 / HO1 / NQO1 pathway on oral cancer cells proliferation, migration, and apoptosis

ObjectiveHerein, we explored the influences of breast cancer susceptibility gene 1 (BRCA1) overexpression on oral cancer cells proliferation, migration, and apoptosis via evaluation of its interactions with nuclear factor erythroid 2-like 2 (NRF2). DesignCAL-27 and DOK cells were transfected with a BRCA1 overexpressing lentivirus. Next, we utilized Western blot and quantitative real-time polymerase chain reaction (qRT-PCR) analyses to evaluate BRCA1, NRF2, and their target gene expressions. Using cell counting kit-8 (CCK-8) assessment, we assessed cell proliferation and a scratch test detected CAL-27 cell migration. Additionally, flow cytometry was employed used to examine cell apoptosis, while an enzyme-linked immunosorbent assa (ELISA) was employed for evaluation of 8-hydroxy-2′- deoxyguanosine (8-OHdG) expression. An immunohistochemical analysis was employed to determine the NRF2 target genes and Ki-67 expressions. ResultsBRCA1 overexpression increased the NRF2 and its target gene transcript and protein expressions. CCK-8 and scratch test results showed that BRCA1 overexpression decreased cell proliferation and weakened CAL-27 cell migratory ability. Flow cytometry results showed that BRCA1 overexpression promoted cell apoptosis in a time-dependent manner, while enzyme-linked immunosorbent assay results showed that BRCA1 overexpression decreased 8-OHdG expression levels in CAL-27 and DOK cells. Immunohistochemical analysis results showed higher expression of NRF2 target genes and Ki-67 in oral squamous cell carcinoma cells. ConclusionsExperiments involving oral cancer cells confirmed that BRCA1 overexpression could up-regulate the NRF2 signalling pathway, reduce oxidative damage, and inhibit cell proliferation and other biological behaviours. The BRCA1 and NRF2 pathways might be associated with oral cancer occurrence and development.

CNPAS2 induced β cell dysfunction by regulating KANK1 expression in type 2 diabetes.

Diabetes mellitus type 2 (T2DM) is formed by defective insulin secretion with the addition of peripheral tissue resistance of insulin action. It has been affecting over 400 million people all over the world. To explore the pathogenesis of T2DM and to develop and implement new prevention and treatment strategies for T2DM. Receiver operating characteristic (ROC) curve analysis was used to conduct diagnostic markers. The expression level of genes was determined by reverse transcription-PCR as well as Western blot. Cell proliferation assays were performed by cell counting kit-8 (CCK-8) tests. At last, T2DM mice underwent Roux-en-Y gastric bypass surgery. We found that NPAS2 was significantly up-regulated in islet β cell apoptosis of T2DM. The ROC curve revealed that NPAS2 was capable of accurately diagnosing T2DM. NPAS2 overexpression did increase the level of KANK1. In addition, the CCK-8 test revealed knocking down NPAS2 and KANK1 increased the proliferation of MIN6 cells. At last, we found that gastric bypass may treat type 2 diabetes by down-regulating NPAS2 and KANK1. This study demonstrated that NPAS2 induced β cell dysfunction by regulating KANK1 expression in type 2 diabetes, and it may be an underlying therapy target of T2DM.

Prognostic model of lung adenocarcinoma based on immunoprognosis-related genes and related drug prediction.

Lung cancer (LC) is the most common malignant tumor in the world, and lung adenocarcinoma (LUAD) is the most common type of LC. Immune microenvironment plays a critical role in cancer from onset to relapse. We aimed to identify an effective immune-related prediction model for assessing prognosis and predicting the relevant tumor therapeutic drugs. According to the RNA sequencing data of LUAD transcriptome in The Cancer Genome Atlas (TCGA) database and the immune-related genes obtained from IMMPORT (The Immunology Database and Analysis Portal) database, immune prognosis-related genes were screened. Weighted gene co-expression network analysis (WGCNA) identified hub genes in differentially expressed immune-related genes (DEIRGs). Least absolute shrinkage and selection operator (LASSO) Cox and ten rounds of cross-validation were used to screen core genes to establish a prognostic model, and in situ hybridization was used to verify the expression of core genes in LUAD. Then the patients from the TCGA database were divided into high-risk group and low-risk group. The survival, tumor microenvironment (TME) and immune cell infiltration of different groups were further analyzed, and the differential genes between the two groups were analyzed by gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Set Enrichment Analysis (GSEA) enrichment analyses. Finally, the small molecular drugs that can inhibit the prognosis of LUAD were screened by Connectivity Map (CMAP), and the therapeutic mechanism of small molecular drug oxibendazole was verified by Cell Counting Kit-8 (CCK-8) experiment. A four-immunoprognosis-related gene model was established to forecast the overall survival (OS) of LUAD through LASSO Cox regression and ten rounds of cross-validation analysis. This prognostic model stratified LUAD patients into low-risk and high-risk groups. According to the findings of the survival analysis, the low-risk group had a greater OS than the high-risk group and the content of immune cells in LUAD was corrected with the survival prognosis of patients. Univariate and multivariate Cox regression also revealed that the prognostic model was an independent prognosis factor in LUAD. Five kinds of small molecular drugs which can inhibit the prognosis of LUAD were screened by CMAP. As shown by CCK-8 test, the small molecular drug "oxibendazole" can effectively inhibit the proliferation of LUAD cells. Based on immune-related prognostic genes, a new prognostic model for LUAD was constructed. Oxibendazole can inhibit the proliferation of LUAD cells, which provides a new idea for the treatment of LUAD.

Construction of a novel tissue engineered meniscus scaffold based on low temperature deposition three-dimenisonal printing technology

To investigate the construction of a novel tissue engineered meniscus scaffold based on low temperature deposition three-dimenisonal (3D) printing technology and evaluate its biocompatibility. The fresh pig meniscus was decellularized by improved physicochemical method to obtain decellularized meniscus matrix homogenate. Gross observation, HE staining, and DAPI staining were used to observe the decellularization effect. Toluidine blue staining, safranin O staining, and sirius red staining were used to evaluate the retention of mucopolysaccharide and collagen. Then, the decellularized meniscus matrix bioink was prepared, and the new tissue engineered meniscus scaffold was prepared by low temperature deposition 3D printing technology. Scanning electron microscopy was used to observe the microstructure. After co-culture with adipose-derived stem cells, the cell compatibility of the scaffolds was observed by cell counting kit 8 (CCK-8), and the cell activity and morphology were observed by dead/live cell staining and cytoskeleton staining. The inflammatory cell infiltration and degradation of the scaffolds were evaluated by subcutaneous experiment in rats. The decellularized meniscus matrix homogenate appeared as a transparent gel. DAPI and histological staining showed that the immunogenic nucleic acids were effectively removed and the active components of mucopolysaccharide and collagen were remained. The new tissue engineered meniscus scaffolds was constructed by low temperature deposition 3D printing technology and it had macroporous-microporous microstructures under scanning electron microscopy. CCK-8 test showed that the scaffolds had good cell compatibility. Dead/live cell staining showed that the scaffold could effectively maintain cell viability (>90%). Cytoskeleton staining showed that the scaffolds were benefit for cell adhesion and spreading. After 1 week of subcutaneous implantation of the scaffolds in rats, there was a mild inflammatory response, but no significant inflammatory response was observed after 3 weeks, and the scaffolds gradually degraded. The novel tissue engineered meniscus scaffold constructed by low temperature deposition 3D printing technology has a graded macroporous-microporous microstructure and good cytocompatibility, which is conducive to cell adhesion and growth, laying the foundation for the in vivo research of tissue engineered meniscus scaffolds in the next step.

Circ-METTL15 stimulates the aggressive behaviors of papillary thyroid cancer cells by coordinating the miR-200c-3p/XIAP axis.

Papillary thyroid carcinoma (PTC) is the most common form of thyroid cancer. The critical importance of circular RNA (circRNA) in a range of cancer types has been lately recognized. However, research on the functions of circRNAs in PTC has been limited thus far. Therefore, this research aimed at exploring the function and mechanism of circ-methyltransferase-like 15 (METTL15) in PTC cells. Quantitative measurements of circ-METTL15, miR-200c-3p, and X-linked inhibitor of apoptosis protein (XIAP) in PTC cells were conducted using reverse transcription-quantitative polymerase chain reaction or Western blot analysis. To investigate cell growth, cell counting kit-8 and colony formation tests were employed, apoptosis was analyzed using flow cytometry, and migration and invasion were studied through Transwell assays. The targeted binding sites between miR-200c-3p and circ-METTL15 or XIAP were predicted by starBase and then verified by dual luciferase reporter assay. circ-METTL15 and XIAP were upregulated in the PTC cells, while miR-200c-3p was downregulated. Downregulating circ-METTL15 or upregulating miR-200c-3p resulted in inhibited proliferation, migration, and invasion of PTC cells, while promoting apoptosis. miR-200c-3p was the downstream molecule of circ-METTL15, and XIAP was the direct target of miR-200c-3p. Forcing XIAP expression obstructed circ-METTL15 silencing to inhibit PTC cell activity. By coopting miR-200c-3p/XIAP, Circ-METTL15 stimulates aggressive behavior in PTC cells.

A novel porous titanium with engineered surface for bone defect repair in load-bearing position.

Porous titanium exhibits low elastic modulus and porous structure is thought to be a promising implant in bone defect repair. However, the bioinert and low mechanical strength of porous titanium have limited its clinical application, especially in load-bearing bone defect repair. Our previous study has reported an infiltration casting and acid corrosion (IC-AC) method to fabricate a novel porous titanium (pTi) with 40% porosity and 0.4 mm pore diameter, which exerts mechanical property matching with cortical bone and interconnected channels. In this study, we introduced a nanoporous coating and incorporated an osteogenic element strontium (Sr) on the surface of porous titanium (named as Sr-micro arch oxidation [MAO]) to improve the osteogenic ability of the pTi by MAO. Better biocompatibility of Sr-MAO was verified by cell adhesion experiment and cell counting kit-8 (CCK-8) test. The in vitro osteogenic-related tests such as immunofluorescence staining, alkaline phosphatase staining and real-time polymerase chain reaction (RT-PCR) demonstrated better osteogenic ability of Sr-MAO. Femoral bone defect repair model was employed to evaluate the osseointegration of samples in vivo. Results of micro-CT scanning, sequential fluorochrome labeling and Van Gieson staining suggested that Sr-MAO showed better in vivo osteogenic ability than other groups. Taking results of both in vitro and in vivo experiment together, this study indicated the Sr-MAO porous titanium could be a promising implant load-bearing bone defect.

Water extract of moschus alleviates erastin-induced ferroptosis by regulating the Keap1/Nrf2 pathway in HT22 cells

Ethnopharmacological relevanceMoschus, first described in the Shennong's Classic of the Materia medicine, is a scarce and precious animal medicine. Modern pharmacological researches have suggested that Moschus has neuroprotective actions, and its mechanism is related to anti-inflammatory, antioxidant, and anti-apoptosis effects. Ferroptosis is one of the major pathologies of Alzheimer's disease (AD) and is widely implicated in the pathogenesis and progression of AD. Although previous studies have suggested that Moschus possesses neuroprotective effect, whether Moschus could mitigate neuronal damages by inhibiting the onset of ferroptosis is unknown in model cells of AD. Aim of the studyThe aim of study was to explore the water extract of Moschus (WEM) on ferroptosis caused by erastin and the potential mechanism. Materials and methodsErastin was used to stimulate HT22 cells to form ferroptosis model to evaluate the anti-ferroptosis effect of WEM by cell counting kit-8 and lactic dehydrogenase (LDH) tests. The malondialdehyde (MDA) and glutathione (GSH) kits are used for detection of MDA and GSH levels, and 2′,7′-dichlorofluorescein diacetate and C11 BODIPY 581/591 fluorescence probe are used for evaluation of reactive oxygen species (ROS) and lipid peroxide (LOOH) levels. And Western blot was used to test nuclear factor erythroid 2-related factor 2 (Nrf2), Kelch-like ECH-associated protein 1 (Keap1), heme oxygenase-1 (HO-1), and ferroptosis associated proteins including glutathione peroxidase 4 (GPX4), cystine/glutamate antiporter subunit (SLC7A11), ferritin heavy chain 1 (FTH1), ferroportin1 (FPN1), transferrin receptor (TFRC). In addition, the Nrf2 inhibitor ML385 was applied to verify whether WEM prevents erastin-induced ferroptosis by activating the Keap1/Nrf2 pathway. ResultsAfter WEM treatment, erastin-induced HT22 cell survival was significantly elevated, the accumulation of intracellular MDA, ROS, and LOOH were significantly reduced, the level of GSH and expressions of ferroptosis inhibitors GPX4 and SLC7A11 were significantly increased, and iron metabolism-related proteins TFRC, FPN1, and FTH1 were regulated. These effects of WEM are implemented by activating the Keap1/Nrf2 pathway. ConclusionsThis study demonstrated that WEM could perform neuroprotective effects by alleviating ferroptosis, verified that WEM treatment of AD can be mediated by the Keap1/Nrf2 pathway, and provided theoretical support for the application of WEM in the treatment of AD.

MiR-128-3p inhibits the proliferation, migration and invasion of human nasopharyngeal carcinoma cells via EMT

Purpose: To investigate the effect of overexpressing miR-128-3p on the migration, invasion, and proliferation of human nasopharyngeal cancer cells, and associated pathways.Methods: Cell counting kit-8 (CCK-8) and clone creation tests were used to investigate the effect of miR-128-3p inhibitor on the growth of C666-1 cells. The inhibitor control and miR-128-3p inhibitor were transfected into human nasopharyngeal cancer cells (C666-1) and allowed to incubate for 48 h. Cell migration and invasion assays were conducted using Transwell and Scratch assays. Cell cycle was assessed using propidium iodide (PI) single-staining method, while expression of miR-128-3p was evaluated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Expression levels of vimentin, E-cadherin, and N-cadherin were determined by Western blot.Results: MiR-128-3p inhibitor transfection in C666-1 cells significantly increased cell proliferation, cell viability, and clonal proliferation, and also significantly reduced miR-128-3p expression levels, and enhanced cell migration and invasion. In C666-1 cells, inhibition of miR-128-3p induced epithelial mesenchymal transition (EMT).Conclusion: MiR-128-3p inhibits nasopharyngeal cancer cells from proliferating, migrating, and invading by regulating epithelial mesenchymal transition. MiR-128-3p may therefore be a viable therapeutic target for nasopharyngeal cancer. Future studies are required to yield pertinent data in support of this hypothesis.

Head and neck squamous cell carcinoma-specific prognostic signature and drug sensitive subtypes based on programmed cell death-related genes.

As a complex group of malignancies, head and neck squamous cell carcinoma (HNSC) is one of the leading causes of cancer mortality. This study aims to establish a reliable clinical classification and gene signature for HNSC prognostic prediction and precision treatments. A consensus clustering analysis was performed to group HNSC patients in The Cancer Genome Atlas (TCGA) database based on genes linked to programmed cell death (PCD). Differentially expressed genes (DEGs) between subtypes were identified using the "limma" R package. The TCGA prognostic signature and PCD-related prognostic genes were found using a least absolute shrinkage and selection operator (LASSO) regression analysis and univariate Cox regression analysis. The robustness of the LASSO analysis was validated using datasets GSE65858 and GSE41613. A cell counting kit-8 (CCK-8) test, Western blot, and real-time reverse transcriptase-polymerase chain reaction (RT-qPCR) were used to evaluate the expression and viability of prognostic genes. Four molecular subtypes were identified in PCD-related genes. Subtype C4 had the best prognosis and the highest immune score, while subtype C1 exhibited the most unfavorable outcomes. Three hundred shared DEGs were identified among the four subtypes, and four prognostic genes (CTLA4, CAMK2N1, PLAU and CALML5) were used to construct a TCGA-HNSC prognostic model. High-risk patients manifested poorer prognosis, more inflammatory pathway enrichment, and lower immune cell infiltration. High-risk patients were more prone to immune escape and were more likely to be resistant to Cisplatin and 5-Fluorouracil. Prognosis prediction was validated in external datasets. The expression of CTLA4, CAMK2N1, PLAU and CALML5 was enhanced in CAL-27 and SCC-25 cell lines, and CALML5 inhibited CAL-27 and SCC-25 cell viability. This study shares novel insights into HNSC classification and provides a reliable PCD-related prognostic signature for prognosis prediction and treatment for patients with HNSC.

Thymoquinone attenuates inflammation in C. Albicans keratitis by activating Nrf2/HO-1 signaling pathway and reducing fungal load

PurposeThis study aims to investigate the anti-inflammatory and antifungal properties of thymoquinone (TQ) and elucidate its mechanism of action in the context of C. albicans keratitis. MethodsVarious methods were employed to identify a safe and effective concentration of TQ with antifungal properties, including the determination of the minimum inhibitory concentration (MIC), the cell counting kit-8 (CCK-8) test, and the Draize experiment. The severity of fungal keratitis (FK) was assessed through clinical ratings and slit-lamp imaging. Fungus burden was determined using plate counting and periodic acid Schiff (PAS) staining. Neutrophil infiltration and activity were investigated through immunofluorescence staining (IFS), myeloperoxidase (MPO) analysis, and hematoxylin and eosin (HE) staining. To explore the anti-inflammatory effects of TQ and its mechanism of action, we employed RT-PCR, ELISA, and western blot techniques. ResultsTQ effectively controlled fungal growth at a concentration of 50 µg/mL while preserving the integrity of mouse corneas. Human corneal epithelial cells (HCECs) remained unaffected by TQ at concentrations ≤ 3.75 µg/mL. Treatment with TQ led to significant improvements in clinical scores, fungal burden, neutrophil infiltration, and the expression of inflammatory factors compared to the DMSO group. Moreover, TQ demonstrated the ability to reduce the levels of inflammatory factors in HCECs stimulated by C. albicans. Additionally, TQ enhanced the expressions of Nrf2 and HO-1 in mouse corneas. The downregulation of cytokines induced by TQ was reversed upon pretreatment with inhibitors of Nrf2 or HO-1. ConclusionTQ exhibits a protective effect in the context of C. albicans keratitis through multiple mechanisms, including inhibition of C. albicans growth, reduction of neutrophil recruitment, activation of the Nrf2/HO-1 pathway, and limitation of the expression of pro-inflammatory factors.

Coix seed oil alleviates synovial angiogenesis through suppressing HIF-1α/VEGF-A signaling pathways via SIRT1 in collagen-induced arthritis rats

BackgroundRheumatoid arthritis (RA) is a chronic autoimmune disorder characterized by symmetric arthritis. Coix Seed Oil (CSO) has been shown to reduce inflammation in collagen induced arthritis (CIA) rats. However, the effect of CSO on synovial angiogenesis in RA is unknown. In this study, we aimed to explore whether CSO could inhibit RA synovial angiogenesis and elucidate the underlying mechanisms.MethodsCIA rat models were established and subjected to different doses of CSO treatments for four weeks in vivo. Arthritis index, paw swelling, and weight were recorded to assess clinical symptoms. Hematoxylin and Eosin staining, Safarnin O fast green staining, Micro-CT, Immunohistochemical, and Immunofluorescence (IF) staining were performed to examined changes in synovial and joint tissues. The serum HIF-1α and VEGF-A levels were evaluated through enzyme-linked immunosorbent assay. Fibroblast-like synoviocytes (FLS) of rats was stimulated with tumor necrosis factor-α (TNF-α) for developing inflammatory model in vitro. Optimal concentrations of CSO and TNF-α for stimulation were measured through Cell Counting Kit-8 test. Wound healing and Transwell migration experiments were employed to determine FLS migratory ability. IF staining was performed to assess HIF-1α nuclear translocation in FLS. Protein levels of SIRT1, HIF-1α, VEGF-A, and CD31 were assessed through Western blot. The isolated aortic rings were induced with recombinant rat VEGF-A 165 (VEGF-A165) to observe the CSO inhibitory impact on angiogenesis ex vivo.ResultsCSO attenuated the progression of arthritis in CIA rats, mitigated histopathological deterioration in synovial and joint tissues, significantly inhibited immature vessels labeled with CD31+/αSMA−, and reduced the micro-vessels in VEGF-A165 induced aortic rings. Moreover, it upregulated SIRT1 protein levels in CIA rats and TNF-α induced FLS, but decreased HIF-1α and VEGF-A protein levels. Furthermore, CSO inhibited the migration ability and HIF-1α nuclear translocation of TNF-α induced FLS. Finally, suppressing SIRT1 levels in TNF-α induced FLS enhanced their migration ability, HIF-1α nuclear translocation, and the protein levels of HIF-1α, VEGF-A, and CD31, whereas the inhibitory effect of CSO on TNF-α induced FLS was severely constrained.ConclusionsThis study indicates that CSO can alleviate synovial angiogenesis through suppressing HIF-1α/VEGF-A signaling pathways via SIRT1 in CIA rats.

Fabrication of epidermal growth factor functionalized polymeric poly(lactic-co-glycolic acid) nanoparticles loaded SN-38 and perfluorocarbon for treatment and care: investigation of antiproliferative effects and apoptosis in cervical cancer cells

Patients with cervical cancer have a significant risk of tumor recurrence and metastasis. The discovery of effective treatments for cervical cancer is urgently needed. Poly(lactic-co-glycolic acid) (PLGA) was recently discovered to offer a promising therapeutic drug carrier. Hence, we developed dual-loaded PLGA nanoparticles (NPs) as a drug carrier to combat this problem with current chemotherapeutic drugs for cervical cancer. We engineered PLGA NPs with epidermal growth factor (EGF) functionalization and co-loaded them with SN-38 and perfluorocarbon (PC) to treat cervical cancer selectively. Cell counting kit-8 test results reveal that newly fabricated NPs effectively induce cell proliferation in cervical cancer cells. Further, flow cytometry, Hoechst 33342 staining and acridine orange and propidium iodide staining were used to determine the apoptosis of SN38/PC@EGF-PLGA NPs in HeLa and CaSki cells. And the release was pH-dependent when tested in vitro. Cervical cancer cells took up targeted SN38/PC@EGF-PLGA NPs at a greater rate than untargeted NPs. Furthermore, HeLa and CaSki cells were more sensitive to apoptosis induction and cell viability suppression when exposed to SN38/PC@EGF-PLGA NPs than nontargeted NPs. The findings of this study improve the exploration of SN38/PC@EGF-PLGA NPs in the new development of effective drug candidates for highly invasive cervical cancer in future.

Long non-coding ribonucleic acid zinc finger E-box binding homeobox 1 antisense RNA 1 regulates myocardial fibrosis in diabetes through the Hippo-Yes-associated protein signaling pathway.

Fibrosis is the principle reason for heart failure in diabetes. Regarding the involvement of long non-coding ribonucleic acid zinc finger E-box binding homeobox 1 antisense 1 (ZEB1-AS1) in diabetic myocardial fibrosis, we explored its specific mechanism. Human cardiac fibroblasts (HCF) were treated with high glucose (HG) and manipulated with plasmid cloning deoxyribonucleic acid 3.1-ZEB1-AS1/microribonucleic acid (miR)-181c-5p mimic/short hairpin RNA specific to sirtuin 1 (sh-SIRT1). ZEB1-AS1, miR-181c-5p expression patterns, cell viability, collagen I and III, α-smooth muscle actin (α-SMA), fibronectin levels and cell migration were assessed by reverse transcription quantitative polymerase chain reaction, cell counting kit-8, western blot and scratch tests. Nuclear/cytosol fractionation assay verified ZEB1-AS1 subcellular localization. The binding sites between ZEB1-AS1 and miR-181c-5p, and between miR-181c-5p and SIRT1 were predicted and verified by Starbase and dual-luciferase assays. The binding of SIRT1 to Yes-associated protein (YAP) and YAP acetylation levels were detected by co-immunoprecipitation. Diabetic mouse models were established. SIRT1, collagen I, collagen III, α-SMA and fibronectin levels, mouse myocardium morphology and collagen deposition were determined by western blot, and hematoxylin-eosin and Masson trichrome staining. Zinc finger E-box binding homeobox 1 antisense 1 was repressed in HG-induced HCFs. ZEB1-AS1 overexpression inhibited HG-induced HCF excessive proliferation, migration and fibrosis, and diminished collagen I, collagen III, α-SMA and fibronectin protein levels in cells. miR-181c-5p had targeted binding sites with ZEB1-AS1 and SIRT1. SIRT1 silencing/miR-181c-5p overexpression abrogated ZEB1-AS1-inhibited HG-induced HCF proliferation, migration and fibrosis. ZEB1-AS1 suppressed HG-induced HCF fibrosis through SIRT1-mediated YAP deacetylation. ZEB1-AS1 and SIRT1 were repressed in diabetic mice, and miR-181c-5p was promoted. ZEB1-AS1 overexpression improved myocardial fibrosis in diabetic mice, and reduced collagen I, collagen III, α-SMA and fibronectin protein levels in myocardial tissues. Long non-coding ribonucleic acid ZEB1-AS1 alleviated myocardial fibrosis through the miR-181c-5p-SIRT1-YAP axis in diabetic mice.

Proteomic profiling of FOLFOX-resistant colorectal cancer cells.

e15588 Background: Chemoresistance is a major cause of colorectal cancer (CRC) mortality. Although multidrug regimen has been established as the backbone of CRC chemotherapy for better treatment efficacy, chemoresistance is common among late-stage CRC. Proteomic profiling of both the chemoresistant and wild-type CRC cells may provide hints for the mechanisms of chemoresistance and suggest targets for improving chemotherapy efficacy. Methods: Human CRC cell lines (DLD-1 and HCT-116) were treated with progressively increased dosage of FOLFOX (5-flurouracil (5-FU), folinic acid (LEU) and oxaliplatin (OXA)) to develop chemoresistance as DLD-1-R and HCT-116-R respectively. After confirmation of the successful development of chemoresistance by Cell Counting Kit-8 test (CCK-8), the comparison of cell samples between chemoresistant and wild-type cell lines were conducted by proteomic profiling using quantitative mass spectrometry (MS)-based techniques. MS and MS/ MS peptides spectra were analysed by Progenesis QI for proteomics software compared with Swiss-Prot dataset. Differentially expressed proteins (DEP) are defined as -1 > log2FC > 1, p < 0.05. Gene-list enrichment by Enrichr (https://maayanlab.cloud/Enrichr/) was employed for identifying the hallmark gene sets of the significantly enriched molecular processes and pathways of each group. All experiments were performed with at least 3 replications. Results: The development of FOLFOX-resistance took one year. The highest drug concentrations achieved were 0.5mM 5-FU, 50µM LEU, and 50µM OXA for DLD-1-R, and 5µM 5-FU, 0.5µM LEU, and 0.5µM OXA for HCT-116-R. The chemoresistance was found to be 14.73 (DLD-1-R) and 1.61 (HCT-116-R) times of the wild-type DLD-1 and HCT-116 respectively from CCK-8 tests. A total of 345 DEP with 154 up-regulated and 191 down-regulated proteins were found in DLD-1-R. While a total of 95 DEP with 66 up-regulated and 29 down-regulated proteins were found in HCT-116-R. Gene ontology molecular process (2021) analyses found that cadherin binding were significantly enriched in both DLD-1-R and HCT-116-R (Adjusted p-value < 0.05). KEGG pathway (2021) analysis found that oxidative phosphorylation, regulation of actin cytoskeleton and HIF-1 signalling pathway are among the significantly enriched pathways in DLD-1-R (Adjusted p value < 0.05). There was no significantly enriched pathway found in HCT-116-R in the same analysis. Conclusions: Our study has successfully established CRC cells with chemoresistance to the FOLFOX regimen. Proteomic profiling has found the cadherin binding as the significantly altered molecular process along with several important signalling pathways in chemoresistant CRC cells. These findings could suggest new directions in the development of anti-chemoresistance strategies of CRC.

MiR-506 targets polypyrimidine tract-binding protein 1 to inhibit airway inflammatory response and remodeling via mediating Wnt/β-catenin signaling pathway.

Airway remodeling, which contributes to the clinical course of childhood asthma, occurs due to airway inflammation and is featured by anomalous biological behaviors of airway smooth muscle cells (ASMCs). microRNA (miRNA) plays an essential role in the etiopathogenesis of asthma. This research was aimed to characterize miR-506 in asthma and uncover potential regulatory machinery. The asthmatic cell model was established by treating ASMCs with transforming growth factor-beta1 (TGF-β1) and assessed by the levels of interleukin (IL)-1β and interferon gamma (IFN-γ). Using real-time quantitative polymerase chain reaction, mRNA expression of miR-506 and polypyrimidine tract-binding protein 1 (PTBP1) was measured. Cell counting kit-8 and Transwell migration tests were used for estimating the capacity of ASMCs to proliferate and migrate. Luciferase reporter assay was used to corroborate whether miR-506 was directly bound to PTBP1. Expression of PTBP1, collagen I and III, and essential proteins of the wingless-related integration (Wnt)/β-catenin pathway (β-catenin, c-MYC and cyclin D1) was accomplished by Western blot analysis. The involvement of Wnt/β-catenin signaling in asthma was confirmed by Wnt signaling pathway inhibitor (IWR-1). miR-506 was poorly expressed in asthmatic tissues and cell model. Functionally, overexpression of miR-506 reduced aberrant proliferation, migration, inflammation and collagen deposition of ASMCs triggered by TGF-β1. Mechanically, miR-506 directly targeted the 3' untranslated region (3-UTR) of PTBP1 and had a negative regulation on PTBP1 expression. Moreover, overexpression of miR-506 suppressed the induction of Wnt/β-catenin pathway. The administration of IWR-1 further validated negative correlation between miR-506 and the Wnt/β-catenin pathway in asthma. Our data indicated that targeting miR-506/PTBP1/Wnt/β-catenin axis might point in a helpful direction for treating asthma in children.

Construction of TRAF6 ubiquitin site 331 mutant colorectal cancer cell stable line and its effect on biological behavior of colorectal cancer cells

Objective: To investigate the effect of ubiquitin mutation at position 331 of tumor necrosis factor receptor related factor 6 (TRAF6) on the biological characteristics of colorectal cancer cells and its mechanism. Methods: lentivirus wild type (pCDH-3×FLAG-TRAF6) and mutation (pCDH-3×FLAG-TRAF6-331mut) of TRAF6 gene expression plasmid with green fluorescent protein tag were used to infect colorectal cancer cells SW480 and HCT116, respectively. The infection was observed by fluorescence microscope, and the expressions of TRAF6 and TRAF6-331mut in cells was detected by western blot. Cell counting kit-8 (CCK-8) and plate cloning test were used to detect the proliferation ability of colorectal cancer cells in TRAF6 group and TRAF6-331mut group, cell scratch test to detect cell migration, Transwell chamber test to detect cell migration and invasion, immunoprecipitation to detect the ubiquitination of TRAF6 and TRAF6-331mut with ubiquitinof lysine binding sites K48 and K63. Western blot was used to detect the effects of TRAF6 and TRAF6-331mut over expression on the nuclear factor kappa-B (NF-κB) and mitogen activated protein kinase mitogen-activated protein kinase (MAPK)/activating protein-1(AP-1) signal pathway. Results: The successful infection of colorectal cancer cells was observed under fluorescence microscope. Western blot detection showed that TRAF6 and TRAF6-331mut were successfully expressed in colorectal cancer cells. The results of CCK-8 assay showed that on the fourth day, the absorbance values of HCT116 and SW480 cells in TRAF6-331mut group were 1.89±0.39 and 1.88±0.24 respectively, which were lower than those in TRAF6 group (2.09±0.12 and 2.17±0.45, P=0.036 and P=0.011, respectively). The results of plate colony formation assay showed that the number of clones of HCT116 and SW480 cells in TRAF6-331mut group was 120±14 and 85±14 respectively, which was lower than those in TRAF6 group (190±21 and 125±13, P=0.001 and P=0.002, respectively). The results of cell scratch test showed that after 48 hours, the percentage of wound healing distance of HCT116 and SW480 cells in TRAF6-331mut group was (31±12)% and (33±14)%, respectively, which was lower than those in TRAF6 group [(43±13)% and (43±7)%, P=0.005 and 0.009, respectively]. The results of Transwell migration assay showed that the migration numbers of HCT116 and SW480 cells in TRAF6-331mut group were significantly lower than those in TRAF6 group (P<0.001 and P<0.002, respectively). The results of Transwell invasion assay showed that the number of membrane penetration of HCT116 and SW480 cells in TRAF6-331mut group was significantly lower than those in TRAF6 group (P=0.008 and P=0.009, respectively). The results of immunoprecipitation detection showed that the ubiquitin protein of K48 chain pulled by TRAF6-331mut was lower than that of wild type TRAF6 in 293T cells co-transfected with K48 (0.57±0.19), and the ubiquitin protein of K63 chain pulled down by TRAF6-331mut in 293T cells co-transfected with K63 was lower than that of wild type TRAF6 (0.89±0.08, P<0.001). Western blot assay showed that the protein expression levels of NF-κB, p-NF-κB and p-AP-1 in TRAF6-331mut-HCT116 cells were 0.63±0.08, 0.42±0.08 and 0.60±0.07 respectively, which were lower than those in TRAF6-HCT116 cells (P=0.002, P<0.001 and P<0.001, respectively). The expression level of AP-1 protein in TRAF6-HCT116 cells was 0.89±0.06, compared with that in TRAF6-HCT116 cells. The difference was not statistically significant (P>0.05). The protein expression levels of NF-κB, p-NF-κB and p-AP-1 in TRAF6-331mut-SW480 cells were 0.50±0.06, 0.51±0.04, 0.48±0.02, respectively, which were lower than those in TRAF6-SW480 cells (all P<0.001). There was no significant difference in AP-1 protein expression between TRAF6-331mut-SW480 cells and TRAF6-SW480 cells. Conclusion: The ubiquitin site mutation of TRAF6 gene at 331 may prevent the binding of TRAF6 and ubiquitin lysine sites K48 and K63, and then affect the expressions of proteins related to downstream NF-κB and MAPK/AP-1 signal pathways, and inhibit the proliferation, migration and invasion of colorectal cancer cells.

Addition of ellagic acid improved the immune ability and delayed the apoptosis of ovarian granulosa cells of Guizhou black goat

Context Follicular development plays an important role in the growth and reproduction of female mammals. Ellagic acid (EA), as a natural antioxidant, has been used in freezing protection of pig semen. However, the effects of EA on immunity and the anti-apoptotic ability of ovarian granulosa cells (GCs) are still unclear. Aims The aim of this study was to analyse the effects of different concentrations of EA on the immune and anti-apoptotic ability of ovarian GCs of Guizhou black goats. Methods In this study, different concentrations of EA (0, 50, 100, 150, 200 μmol/L) were added to the culture of ovarian GCs in vitro, and Cell-Counting Kit 8 (CCK8) assay, cell wound scratch assay, and real-time fluorescence quantitative polymerase chain reaction (RT–qPCR) assay were used to detect the effects of different concentrations of EA on the proliferation, migration, and reproductive marker genes of ovarian GCs. Then the optimal addition concentration of EA was selected and the effects of EA supplementation on immune factors, cytochrome P450 family 19 subfamily A member 1 gene (CYP19A1), estradiol concentrations, intracellular reactive oxygen species concentrations, and apoptosis-related protein expression were detected by RT–qPCR, enzyme-linked immunosorbent assay (ELISA), ROS, and western blotting on the basis of the optimal addition concentration. Key results The CCK8 test and cell scratch test showed that the addition of EA could significantly inhibit the proliferation and migration ability of ovarian GCs compared with the control group, and a dose effect was observed with the increase in concentration. RT–qPCR results showed that different concentrations of EA significantly increased the expression of genes associated with reproduction, including bone morphogenetic protein 15 (BMP15), bone morphogenetic protein receptor 1B (BMPR-1B), growth differentiation fFactor 9 (GDF9), and follicle-stimulating hormone β subunit (FSHβ), and the maximum increase was observed at 150 μmol/L EA. Further analyses using 150 μmol/L EA as the optimal concentration showed significantly increased expressions of CYP19A1, interleukin-10 (IL-10), and superoxide dismutase (SOD2) after EA supplementation, while the expression of IL-8 was significantly decreased compared with those of the control group. ELISA and ROS showed that both intracellular and extracellular estradiol concentrations were higher, while ROS concentrations were significantly lower than those in the control group. Western blotting results showed that 150 μmol/L EA significantly decreased the expression of Caspase-3 and Caspase-9 and the ratio of BCL2-associated X:B-cell lymphoma-2. Conclusions The supplementation of 150 μmol/L EA had significant effects on improving GC immunity and delaying GC apoptosis in goats. The addition of EA also increased the expression of BMP15, BMPR-1B, GDF9, FSHβ, and CYP19A1 and promoted the secretion of estradiol in GCs. Implications These results provided a preliminary lead for further research on the effect of EA on the maturation and development of goat oocytes in vitro.

Downregulation of MACC1 facilitates the reversal effect of verapamil on the chemoresistance to active metabolite of irinotecan in human colon cancer cells

The aim of this study is to investigate the reversal effect of verapamil (VER) on chemoresistance to irinotecan (CPT-11) in human colon cancer cells and relevant mechanisms. Cell counting kit-8 (CCK-8) test and colony-forming unit (CFU) experiment results show that VER strengthens the sensitivity of human colon cancer cell line HT29 to CPT-11 but has a small effect on SW480 cells. High-throughput transcriptome sequencing, RT-PCR, and Western blot results show that the inhibition of metastasis-associated in colon cancer-1 (MACC1) expression by VER is the key factor for reversal effect on chemoresistance to CPT-11. Transfection experiments further show that VER can reverse the resistance of human colon cancer cells to SN-38, the active metabolite of CPT-11, when MACC1 is overexpressed. The nude mouse transplantation tumor experiment provides an in vivo proof that VER can strengthen sensitivity to CPT-11 in drug-resistant human colon cancer cells, and the effect might be related to the inhibited expression of MACC1. In summary, VER might strengthen the reversal effect of VER on chemoresistance to CPT-11 in human colon cancer cells and facilitate the apoptosis of human colon cancer cells by downregulating MACC1 expression.

Effects of metformin on Sonic hedgehog subgroup medulloblastoma progression: In vitro and in vivo studies.

Metformin is a first-line drug for type 2 diabetes, and its anticancer effects have also been widely studied in recent years. The Sonic hedgehog (Shh) signaling pathway is involved in the initiation and progression of medulloblastoma. In order to develop a new treatment strategy for medulloblastoma (MB), this study investigated the inhibitory effect of metformin on MB and the underlying mechanism of metformin on the Shh signaling pathway. The effect of metformin on proliferation was evaluated by the cell counting kit-8 (CCK-8) test and colony formation experiment. The effect of metformin on metastasis was assessed by the scratch-wound assay and transwell invasion assay. Cell cycle and apoptosis were evaluated by flow cytometry, and the associated proteins were examined by western blotting. The mRNA and protein expression levels related to the Shh pathway were measured by quantitative PCR, western blotting, and immunofluorescence staining. The xenograft murine model was carried out to evaluate the anticancer effect of metformin on medulloblastoma in vivo. Metformin inhibited proliferation and metastasis of the Shh subgroup MB cell line, and the inhibitory effect on proliferation was related to apoptosis and the block of the cell cycle at the G0/G1 phase. Animal experiments showed that metformin inhibits medulloblastoma growth in vivo. Moreover, metformin decreased mRNA and protein expression levels of the Shh pathway, and this effect was reversed by the AMP-activated protein kinase (AMPK) siRNA. Furthermore, the pro-apoptotic and cell cycle arrest effects of metformin on Daoy cells could be reversed by the Shh pathway activators. Our findings demonstrated that metformin could inhibit medulloblastoma progression in vitro and in vivo, and this effect was associated with AMPK-mediated inhibition of the Shh signaling pathway in vitro studies.

Mechanism of LncHOTAIR Regulating Proliferation, Apoptosis, and Autophagy of Lymphoma Cells through hsa-miR-6511b-5p/ATG7 Axis.

Objective To explore the role of LncHOTAIR in apoptosis and autophagy in lymphoma. Methods The interaction between LncHOTAIR and miR-6511b-5p, as well as between miR-6511b-5p and ATG7, was verified by a dual luciferase assay. LncHOTAIR overexpression lentivirus was transducted and siATG7s were transfected into Raji and BJAB lymphoma cells, and the efficiency was verified by qPCR. Lymphocyte proliferation was detected by the cell counting kit-8 (CCK8) test, and autophagy was detected by transmission electron microscopy. The protein expressions of ULK1, Beclin1, ATG7, LC3, Bax, cleaved-caspase 3, and Bcl-2 were detected using Western blots. Results There was a targeting relationship between LncHOTAIR and miR-6511b-5p and between miR-6511b-5p and ATG7. LncHOTAIR overexpression promoted the proliferation and autophagy of Raji and BJAB cells, significantly upregulated ATG7, Beclin1, ULK1, Bcl-2, and LC3-II/LC3-I levels, and downregulated Bax and cleaved-caspase3 levels. siATG7 significantly inhibited the proliferation and autophagy of Raji and BJAB cells and promoted their apoptosis. Conclusion LncHOTAIR/hsa-miR-6511b-5p/ATG7 could regulate the proliferation, apoptosis, and autophagy of Raji and BJAB lymphoma cells.