The aim of the present study was to determine the effects of the Ca2+/calmodulin-dependent protein kinase pathway inhibitor KN93 on osteoclastogenesis. RAW264.7 cells were incubated with macrophage colony-stimulating factor (M-CSF) + receptor activator of nuclear factor kappa-light-chain-enhancer of activated B cells ligand (RANKL) to stimulate osteoclastogenesis and then treated with 10 mu MKN93. The methods included tartrate-resistant acid phosphatase (TRAP) staining, bone resorption activity assays, filamentous (F)-actin staining, determination of intracellular calcium ([Ca2+](i)) levels, monitoring of osteoclast-specific gene expression levels and measurement of key transcription factors protein levels. The results suggested that KN93 inhibited the formation of TRAP-positive multinucleated cells, shaping of F-actin rings and resorption activity of the cells. In addition, KN93 decreased the concentration of [Ca2+], expression levels of osteoclast specific genes and protein levels of critical transcription factors in the M-CSF + RANKL-induced osteodast model. In summary, KN93 may directly affect the differentiation and activation of ostcoclasts, potentially through the Ca2+/calmodulin-dependent protein kinase signaling pathway.
The activation of Notch signalling induces epithelial-mesenchymal transition (EMT), but this signalling pathway and its association with EMT in the context of cell motility in oral squamous cell carcinoma (OSCC) remains unclear. The present study aimed to investigate the role of the Notch signalling pathway and EMT in the metastatic potential of OSCC using 2 cell lines, Tca8113 and CAL27. The data demonstrated that zinc finger domain SNAI1 (Snail) knockdown by small interfering RNA decreased the expression of vimentin and increased the expression of epithelial cadherin (E-cadherin). In addition, silencing Snail also significantly inhibited cell migration in the 2 OSCC cell lines. It was also identified that blocking Notch signalling with the g-secretase inhibitor DAFT decreased the expression of the EMT markers Snail and vimentin and increased E-cadherin expression, accompanied by a significant inhibition of cell migration in the 2 OSCC cell lines. These data dearly indicate that Notch signalling mediates EMT to promote metastasis in OSCC cells. Therefore, targeting Notch signalling and its association with EMT may provide novel insights into the mechanism of invasion and metastasis in OSCC and potential therapeutic interventions.
Expression efficacy of recombinant protein in current expression systems is generally low. Therefore, the expression levels of recombinant proteins in the breast milk of transgenic animals are typically low. In view of this, the present study aimed to construct homozygous transgenic rabbits with a high expression level of recombinant human plasminogen activator (rhPA) during the entire lactation period. Homozygous transgenic rabbits were obtained using an effective rhPA mammary-specific expression vector PCL25/rhPA. The expression level and thrombolytic ability of rhPA in the milk of both homozygous and hemizygous rabbits were detected by enzyme-linked immunosorbent and fibrin agarose plate assays. It was observed that the expression of rhPA was constant during the entire lactation period in homozygous rabbits, while the expression of rhPA declined slowly in hemizygote rhPA transgenic rabbits during the lactation period. In addition, the expression of rhPA in homozygous transgenic rabbit was similar to 950 mu g/ml, which was markedly higher in comparison with that in hemizygote rabbits. Furthermore, increased gene copy number was observed to increase the expression level of rhPA at the same integration vector.
Cervical cancer is one of the most common types of female malignant tumor. It is well established that radiotherapy (RT) is the first-line treatment of cervical cancer; however, radioresistance is a substantial obstacle to cervical cancer RT. At present, the mechanism underlying radioresistance remains unclear. Emerging evidence has demonstrated that long non-coding RNAs (lncRNAs) function as crucial regulators of diverse cancers. Aerobic glycolysis, which is a common phenomenon in cancer cells, is associated with various biological functions, including radioresistance. To the best of our knowledge, the present study is the first to explore the role of the lncRNA urothelial cancer associated 1 (UCA1) in cervical cancer radioresistance. In the present study, irradiation was used to establish irradiation-resistant (IRR) cells, after which a clonogenic survival assay was used to validate radioresistance, reverse transcription-quantitative polymerase chain reaction was used to evaluate the expression levels of UCA1 and western blotting was conducted to detect the expression levels of glycolysis-related proteins. In addition, a glucose/lactate assay kit was used to evaluate glucose/lactate concentrations and cells were transfected with small interfering RNA/pcDNA to regulate the expression of UCA1. Following the establishment of IRR cell lines (SiHa-IRR and HeLa-IRR), it was demonstrated that SiHa-IRR and HeLa-IRR cells exhibited increased expression levels of UCA1 and enhanced glycolysis. Dysregulation of UCA1 and inhibition of glycolysis affected radioresistance of cervical cancer cells. In addition, the results indicated that UCAI promoted radioresistance-associated glycolysis in SiHa-IRR and HeLa-IRR cells, with the enzyme hexokinase 2 (HK2) acting as a significant regulator in this process. Inhibiting glycolysis by 2-DG reversed the effects of UCAI overexpression on HK2 protein expression and radioresistance in SiHa and HeLa cells. Taken together, these findings suggested that UCA1 may have an important role in regulating radioresistance through the HK2/glycolytic pathway, providing novel potential targets to improve cervical cancer RT.
The present study aimed to identify the molecular basis of the arthrogryposis-renal dysfunction-cholestasis (ARC) syndrome, which is caused by mutations in the vacuolar protein sorting 33 homolog B (VPS33B) gene. The microarray dataset GSE83192, which contained six liver tissue samples from VPS33B knockout mice and four liver tissue samples from control mice, was downloaded from the Gene Expression Omnibus database. The differentially expressed genes (DEGs) were screened by the Limma package in R software. The DEGs most relevant to ARC were selected via weighted gene co-expression network analysis to construct a protein-protein interaction (PPI) network. In addition, module analysis was performed for the PPI network using the Molecular Complex Detection function. Functional and pathway enrichment analyses were also performed for DEGs in the PPI network. Potential drugs for ARC treatment were predicted using the Connectivity Map database. In total, 768 upregulated and 379 downregulated DEGs were detected in the VPS33B knockout mice, while three modules were identified from the PPI network constructed. The DEGs in module 1 (CD83, IL1B and TLR2) were mainly involved in the positive regulation of cytokine production and the Toll-like receptor (TLR) signaling pathway. The DEGs in module 2 (COL1A1 and COL1A2) were significantly enriched with respect to cellular component organization, extracellular matrix-receptor interactions and focal adhesion. The DEGs in module 3 (ABCG8 and ABCG3) were clearly associated with sterol absorption and transport. Furthermore, mercaptopurine was identified to be a potential drug (connectivity score=-0.939) for ARC treatment.In conclusion, the results of the current study may help to further understand the pathology of ARC, and the DEGs identified in these modules may serve as therapeutic targets.
Although melatonin has been shown to exert marked antitumor effects against a variety of cancers, the underlying mechanisms remain to be fully elucidated. It has been hypothesized that the anticancer properties of melatonin are associated with its ability to suppress epithelial-to-mesenchymal transition (EMT) of cancer cells. In the present study, melatonin effectively suppressed interleukin (IL)-1 beta-induced EMT in human gastric adenocarcinoma (GA) cells. Sequential treatment of GA cells with melatonin after IL-1 beta challenge markedly reversed the IL-1 beta-induced morphological changes, reduced cell invasion and migration, increased beta-catenin and E-cadherin expression, and downregulated fibronectin, vimentin, Snail, matrix metalloproteinase (MMP)2 and MMP9 expression. Moreover, IL-1 beta-induced activation of NF-kappa B was attenuated following treatment with melatonin. Knockdown of NF-kappa B significantly reduced the IL-1 beta-induced EMT in GA cells. Taken together, these findings indicate that melatonin may act by suppressing EMT and tumor progression by inhibiting NF-kappa B activity.
Numerous studies have demonstrated the presence of resident nucleus pulposus stem/progenitor cells (NPSCs) in the tissue of the intervertebral disc (IVD). However, the cellular identity of NPSCs during IVD degeneration and ageing are poorly defined at present, despite significant progress in the understanding of NPSC biology. In the present study, NPSCs were isolated from human degenerated IVD and were characterized by flow cytometry, gene expression assays and proliferation and multipotency analysis. The results of the present study demonstrated that NPSCs isolated from human degenerated IVD may be divided into two groups according to the expression of mesenchymal stern cell (MSC) surface markers: The high expression of MSC surface markers group (H-NPSCs) was highly positive for CD29, CD44, CD73, CD90 and CD105 at rates >95%, and the low expression of MSC markers surface markers group (L-NPSCs), with the expression of CD29 and CD105 exhibiting individual variability, however, all at rates <95%. The donors for H-NPSCs were aged <20 years, while the majority of donors for L-NPSCs were aged >25 years, with one exception aged <20 years. The results highlighted that the low expression of MSC surface markers in NPSCs from aged and degenerated NP tissues were associated with a low rate of proliferation and reduced differentiation potential, as well as downregulation of the NP progenitor marker Tie2 and higher expression of NP cell-specific markers. These findings demonstrated that the regenerative potential of human NPSCs declines with ageing and degeneration of the IVD.
Stem cell transplantation is a promising clinical strategy for curing ischemic cardiomyopathy. However, its efficacy is impaired by low cell survival following transplantation, partly caused by insufficient resistance of the transplanted stern cells to severe oxidative stress at the injury site. In the current study, it was demonstrated that the small-molecule macrophage migration inhibitory factor (MIF) enhanced the defense of bone marrow-derived mesenchymal stem cells (MSCs) against hypoxia/serum deprivation (SD)-induced apoptosis in vitro. MIF significantly suppressed apoptosis and caspase family activities through inhibition of long intergenic noncoding (line) RNA-p21 to maintain activation of the Wnt/beta-catenin signaling pathway. The regulatory loop between MIF and the lincRNA-p21-Wnt/beta-catenin signaling pathway was identified to be associated with the inhibition of oxidative stress. The involvement of the lincRNA-p21-Wnt/beta-catenin signaling pathway in the effects of MR; in NISCs by overexpression of lincRNA-p21and silencing p-catenin using small interfering RNA was also demonstrated, both of which abolished the anti-apoptotic and anti-oxidative effects of MIF in MSCs under hypoxia/SD conditions. In conclusion, MIF protected MSCs from hypoxia/SD-induced apoptosis by interacting with lincRNA-p21, leading to activation of the downstream Wnt/beta-catenin signaling pathway and decreased oxidative stress. Thus, treatment with MIF may have important therapeutic implications in improving MSC survival and therapeutic efficiency.
Aerobic glycolysis is one of the characteristics of tumor metabolism and contributes to the development of tumors. Studies have identified that microRNA (miRNA/miR) serves an important role in glucose metabolism of tumors. miR-199a-3p is a member of the miR-199a family that controls the outcomes of cell survival and death processes, and previous studies have indicated that the expression of miR-199a-3p is low and may be an inhibitor in several cancer types, including testicular tumors. The present study discussed the role and underlying mechanism of miR-199a-3p in aerobic glycolysis of Ntera-2 cells and identified its downstream factors. Firstly, miR-199a-3p exhibited an inhibitory effect on lactic acid production, glucose intake, and reactive oxygen species (ROS) and adenosine 5'-triphosphate (ATP) levels in Ntera-2 cells. Then, using bioinlbrmatics, recombinant construction and a dual luciferase reporter gene system, transcription factor Specificity protein 1 (Sp1) was determined as the direct target of miR-199a-3p. Also, downregulation of Spl by RNA interference decreased lactic acid production, glucose intake, and ROS and ATP levels in Ntera-2 cells. Subsequently, through a functional rescue experiment, it was identified that the overexpression of Spl may abate the inhibition of miR-199a-3p on glucose metabolism, with the exception of ATP level, suggesting a reciprocal association between Spl and miR-199a-3p. Finally, it was determined that miR-199a-3p overexpression and Spl knockdown decreased lactate dehydrogenase A (LDHA) protein expression, which indicated that LDHA is a downstream target of the miR-199a-3p/Sp1 signaling pathway. To additionally verify the regulation of LDHA expression by 199a-3p/Sp1, a LDHA promoter reporter plasmid was generated and the high activity of the promoter, which contained 3 potential Spl binding elements, was confirmed. In addition, the overexpression of Spl led to the increased activity of the LDHA promoter, whereas knockdown of Spl exhibited the opposite effect. Therefore, the results of the present study demonstrated that miR-199a-3p can inhibit LDHA expression by downregulating Spl, and provided mechanistic evidence supporting the existence of a novel miR-199a-3p/Sp1/LDHA axis and its critical contribution to aerobic glycolysis in testicular cancer cells.
The present study investigated the effects of formononetin (FMN) against balloon injury-induced neointima formation in vivo and platelet-derived growth factor (PDGF)-BB-induced proliferation and migration of vascular smooth muscle cells (VSMCs) in vitro, and explored the underlying mechanisms. A rat model of carotid artery injury was established, in order to examine the effects of FMN on balloon injury-induced neointima formation. Histological observation of the carotid artery tissues was conducted by hematoxylin and eosin staining. VSMC proliferation during neointima formation was observed by proliferating cell nuclear antigen staining. Subsequently, rat aortic VSMCs were isolated, and the effects of FMN on PDGF-BB-induced VSMC proliferation and migration were determined using Cell Counting Kit-8 and Transwell/wound healing assays, respectively. Immunohistochemical and immunocytochem la staining was applied to measure the expression of transforming growth factor (TGF)-beta in carotid artery tissues and VSMCs, respectively. SMAD family member 3 (Smad3)/phosphorylated (p)-Smad3 expression was examined by western blotting. FMN treatment significantly inhibited the abnormal proliferation of smooth muscle cells in neointima, and alterations to the vascular structure were attenuated. In addition, pretreatment with FMN effectively inhibited the proliferation of PDGF-BB-stimulate VSMCs (P<0.05). FMN also reduced the number of cells that migrated to the lower surface of the Transwell chamber and decreased wound-healing percentage (P<0.05). The expression levels of TGF-beta were decreased by FMN treatment in vivo and in vitro, and Smad3/p-Smad3 expression was also markedly inhibited. In conclusion, FMN significantly protected against balloon injury-induced neointima formation in the carotid artery of a rat model; this effect may be associated with the regulation of VSMC proliferation and migration through altered TGF-beta 1/Smad3 signaling.
In the present study, the role and mechanism of microRNA-634 (miRNA-634) in the adjustment of nerve inflammation and apoptosis in cerebral infarction were investigated. In a cerebral infarction rat model, the expression of miRNA-634 was increased, compared with that in the normal control group. The upregulated expression of miRNA-634 in an in vitro model of cerebral infarction increased cell apoptosis and the protein expression of capsase-3/B-cell lymphoma 2-associated X protein (Bax) via inactivation of the phosphoinositide 3-kinase (PI3K)/Akt pathway. The downregulation of miRNA-634 enhanced cell growth and inhibited cell apoptosis in the in vitro model of cerebral infarction through induction of the PI3K/Akt pathway. Subsequently, a PI3K inhibitor was used to inhibit the expression of PI3K in the in vitro model of cerebral infarction via the downregulation of miRNA-634, which showed that cell apoptosis and the protein expression of capsase-3/Bax were also increased. A PI3K. agonist reduced the effects of the upregulation of miRNA-634 in the in vitro model of cerebral infarction. In conclusion, the data obtained demonstrated the possible future use of miRNA-634 as a therapeutic target in cerebral infarction through the PI3K/Akt pathway.
The drug resistance of cancer remains a major obstacle to successful chemotherapy. New strategies for improving chemotherapeutic efficacy arc urgently required. Recent studies have indicated that LIPC plays a role in promoting the liver metastasis of colorectal cancer. In the present study, we aimed to investigate the effects of LIPC on theproliferation and clone formation of colorectal cancer-derived cells, and chemoresistance in hepatoblastoma-derived HepG2 cells. The activity and expression of UPC were determined in the cell lines by RT-qPCR and western blot analysis. HepG2 cells in which LIPC was knocked down by UPC short hairpin RNA (shRNA) and control cells [shRNA control (shCON)] were established and analyzed for cell proliferation and colony formation rates. FACS analysis was used to explore the association between LIPC and the tumor-derived cell biomarker, CD133, and the percentages of CD133-positive cells were assessed by FACS. Additionally, shLIPC- and shCON-transfected cells were treated with various concentrations of doxorubicin and 5-floxuridine (5-FU), and cell viability was determined by MTT assay. mRNA levels in the shLIPC- and shCON-transfected cells were compared by cDNA microarray and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. The results revealed that the HepG2 cells exhibited a relatively higher LIPC activity and expression levels compared to the other colon cancer cell lines. The downregulation of LIPC in the HepG2 cells was associated with the decreased expression of CD133, decreased cell proliferation and colony formation, as well as increased resistance to chemotherapy. KEGG analysis of the cDNA microarray data revealed increased levels in the cell adhesion molecule (CAM) pathway, including CLDN10 and CLDN1, indicating that CAMs may play a role in LIPC-mediated tumor progression. The present findings indicate a potential role of LIPC as a promising therapeutic target in cancer.
Intestinal injury is a common complication following intracerebral hemorrhage (ICH), which leads to malnutrition, impaired immunity and unsatisfactory prognosis. Previous studies have revealed the pathogenesis of intestinal injury following traumatic brain injury using ischemic stroke models. However, the effects of ICH on intestinal injury remain unknown. The present study aimed to investigate the pathological alterations and molecular mechanism, as well as the time course of intestinal injury following ICH in mice. Male C57BL/6 mice were randomly divided into the following seven groups (n=6 mice/group): Control group, which underwent a sham operation, and six ICH groups (2, 6, 12 and 24 h, and days 3 and 7). The ICH model was induced by stereotactically injecting autologous blood in two stages into the brain. Subsequently, intestinal tissue was stained with hematoxylin and eosin for histopathological examination. Small intestinal motility was measured by charcoal meal test, and gut barrier dysfunction was evaluated by detecting the plasma levels of endotoxin. Quantitative polymerase chain reaction (qPCR), immunohisto-chemistry and ELISA analysis were performed to evaluate the mRNA and protein expression levels of inflammatory cytokines [interleukin (IL)-1 beta, IL-6, tumor necrosis factor-alpha, intercellular adhesion molecule 1, monocyte chemotactic protein 1 and chemokine (C-C motif) ligand-5] in intestinal tissue and serum. Furthermore, intestinal leukocyte infiltration was detected by measuring myeloperoxidase activity. Oxidative stress was indirectly detected by measuring reactive oxygen species-associated markers (malondialdehyde content and superoxide dismutase activity assays) and the mRNA and protein expression levels of antioxidant genes [nuclear factor (erythroid-derived 2)-like 2, manganese superoxide dismutase and heme oxygenase 1] by qPCR and western blot analysis. The results demonstrated that significant destruction of the gut mucosa, delayed small intestinal motility, intestinal barrier dysfunction, and increased inflammatory responses and oxidative stress occurred rapidly in response to ICH. These symptoms occurred as early as 2 h after ICH and persisted for 7 days. These findings suggested that ICH may induce immediate and persistent damage to gut structure and barrier function, which may be associated with upregulalion of inflammation and oxidative stress markers.
Periodontitis can exert a severe impact on the life of patients, and the use of stem cell therapy for this disease is promising. The inflammatory response consequent to periodontitis can promote stem cell proliferation. Activated inflammation triggers inhibitory cytokinc secretion, thus reducing inflammation subsequent to stem cell activation. High-fluence low-power laser irradiation (HF-LPLI) has the ability to regulate stem cell function through its effect on inflammation. Thus, the aim of the present study was to examine whether HF-LPLI is able to activate stem cells to promote regeneration in periodontitis by promoting inflammation resolution, as well as to evaluate the underlying mechanism of action if an effect is observed. Stem cells were treated with HF-LPLI following inflammation activation. Reverse transcription-quantitative polymerase chain reaction and EdU assay were used to evaluate cell proliferation and differentiation. Flow cytometry and immunofluorescence were also used to detect the ability of HF-LPLI to regulate the surrounding inflammatory environment. Animal models of periodontal disease were treated with stem cells and HF-LPLI, and regeneration was detected by hematoxylin and eosin staining and in vivo imaging. It was observed that HF-LPLI promoted inflammation resolution by reducing the excessive inflammatory response, and finally stimulated stem cell proliferation and differentiation. Furthermore, in vivo results revealed that stem cells treated with HF-LPLI induced bone regeneration. HF-LPLI stimulated stem cell proliferation and differentiation by promoting inflammation resolution subsequent to stein cell activation, providing a new strategy for the clinical treatment of periodontitis.
The inhibition of proprotein convertase subtilisin/kexin type 9 (PCSK9) protects a variety of cell types against neuronal apoptosis by binding to apolipoprotein E receptor 2 (ApoER2). The present study aimed to determine the association between PCSK9/ApoER2 signaling and neuronal apoptosis following middle cerebral artery occlusion (MCAO) injury in hyperlipidemic mice. For this purpose, C57BL/6 mice fed with a high-fat diet (HFD) for 6 weeks were exposed to NICAO. Subsequently, PCSK9 was inhibited by a lentiviral vector harboring short-hairpin RNA (shRNA) targeting PCSK9, which was stereotaxically injected into the cerebral cortex of mice. At 48 h post-ischemia, hematoxylin-eosin staining and a terminal deoxynucleotidyl transferase dUTP nick end labeling assay were performed to determine cerebral tissue injury and apoptosis. PCSK9 and ApoER2 expression levels were assessed by reverse transcription-quantitative polymerase chain reaction, immunohistochemistry and western blotting. The results indicated that hyperlipidemia and increased PCSK9 expression were evident in RFD mice. Cerebral histological injury and neuronal apoptosis, as well as PCSK9 and ApoER2 levels, which were increased upon ischemia in hyperlipidemic mice, were attenuated by PCSK9 shRNA treatment. These protective effects of PCSK9 shRNA interference were associated with decreased neuronal apoptosis and a reduced level of ApoER2 expression in the hippocampus and cortex. The data of the present study demonstrated that the PCSK9 shRNA-mediated anti-apoptotic effect induced by MCAO in hyperlipidemic mice is associated with ApoER2 downregulation, which may be a potential new therapy for stroke treatment in patients with hyperlipidemia.