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MicroRNAs (miRNAs/miRs) are non-coding RNAs that regulate protein synthesis by targeting mRNAs for translational repression or degradation. Previous studies have reported that aberrant expression of miR‑744 may be involved in human osteosarcoma; however, the underlying mechanisms remain elusive. In the present study, the expression levels of miR‑744 and its downstream signals were determined by reverse transcription‑quantitative PCR and western blotting. Cell proliferation was assessed using the bromodeoxyuridine assay, and the target of miR‑744 was investigated using a dual‑luciferase activity assay. The present study identified a significant upregulation of miR‑744 in osteosarcoma tissues compared with adjacent non‑tumor tissues. Furthermore, it was demonstrated that ectopic overexpression of miR‑744 induced by a miR‑744 precursor significantly enhanced proliferation of the osteosarcoma cell line MG63, whereas opposite results were observed following suppression of miR‑744 with its inhibitor. Moreover, as a unique anti‑oncogene, PTEN was identified as a direct target of miR‑744. It was confirmed that miR‑744 downregulated PTEN expression in MG63 cells by targeting the PTEN 3'untranslated region, and that the downstream AKT signal was also regulated by miR‑744. Collectively, the present results suggested that miR‑744 promoted proliferation of human osteosarcoma cells by directly regulating the PTEN/AKT signaling pathway.

Club cell protein (CC16) is expressed primarily by club cells possesses anti‑inflammatory properties and is located in the bronchiolar epithelium. Previous studies have demonstrated that CC16 deficiency is associated with the progression of chronic obstructive pulmonary disease (COPD). In the present study, the therapeutic effects of recombinant rat CC16 protein in mice with COPD were examined and the underlying mechanisms investigated. A total of 30 adult male C57/BL6 mice were randomly divided into three groups (10 mice/group). A mouse COPD model was generated by exposing 20 mice to cigarette smoke (CS) for 24 weeks. A total of 10 mice were treated intranasally with rCC16 (2.5 µg/g body weight) and control mice were exposed to normal room air. Results indicated that rCC16 treatment ameliorated pathological damage in the lungs and reduced the production of tumor necrosis factor (TNF)‑α, interleukin (IL)‑6 and IL‑8, which were induced by CS exposure. After rCC16 administration, endogenous CC16 was upregulated and the body weight of COPD mice was increased, whereas the opposite was observed in CS‑exposed mice. Additionally, rCC16 treatment inhibited the DNA binding of NF‑κB/p65 in lung tissues and reduced nuclear translocation of NF‑κB/p65 in BALF and epithelial cells. Moreover, rCC16 treatment lead to a decrease in the total number of BALF cells, including macrophages, which was elevated in COPD mice. In conclusion, the present results demonstrate that rCC16 has therapeutic effects on COPD by downregulating pro‑inflammatory factors via the NF‑κB pathway.

Platelet-activating factor (PAF) promotes glomerular extracellular matrix (ECM) deposition, primarily through activation of the protein kinase C (PKC) pathway. The present study was designed to investigate whether atorvastatin, which mediates a protective effect against glomerular ECM deposition and diabetic neuropathy, may interfere with the PKC-transforming growth factor-β1 (TGF-β1) pathway in a model of human mesangial cells (HMCs) exposed to a high glucose (HG) and lysophosphatidylcholine (LPC) environment. HMCs were divided into three treatment groups: Control, high glucose and lysophosphatidylcholine (HG+LPC), and HG+LPC+atorvastatin. Cells were cultured for 24 h. The levels of the ECM-associated molecules collagen IV (Col IV) and fibronectin (Fn) in the supernatant were detected using an ELISA kit. PKC-β1, TGF-β1 and PAF-receptor gene expression was detected by reverse transcription-quantitative polymerase chain reaction. PKC-β1 and TGF-β1 protein expression was detected by western blotting, and the subcellular localization of PKC-β1 was assessed using immunofluorescence. The results indicated that atorvastatin may reduce the secretion of ECM components (Fn and Col IV) in HMCs in a HG and LPC environment, by inhibiting the increase in PAF secretion and the activation of the PKC-TGF-β1 signaling pathway.

Dexamethasone (Dex) and other glucocorticoids are widely used to treat serious infections and immunological diseases, however they may cause steroid‑induced avascular necrosis of the femoral head (SANFH). Salidroside (Sal) has demonstrated an anti‑apoptotic effect on neurocytes by activating the phosphoinositide 3‑kinase (PI3K)/protein kinase B (Akt) signaling pathway. In the present study, primary osteoblasts were used in vitro and in rats in vivo to determine the anti-apoptotic effect of Sal on SANFH. The result of the present study demonstrated that pretreatment with Sal increased the cell survival rate while decreasing the cell apoptosis and lactate dehydrogenase release rate. Additionally, Sal also caused the reduction of TUNEL positive cells in TUNEL staining assay. Sal decreased the expression of cleaved caspase-3, cleaved caspase‑9, apoptosis regulator BAX and cytochrome C, while it increased the expression of B cell lymphoma‑2 and phosphorylated‑Akt in Dex‑induced osteoblasts. In vivo Sal protected against SANFH in rats by decreasing the percentage of empty lacunae. The present study demonstrated that Sal alleviated Dex‑induced osteoblast apoptosis by activating the PI3K/Akt signaling pathway and downregulating caspase‑3 expression in osteoblasts. Sal also protected against SANFH in a rat model of SANFH by decreasing the percentage of empty lacunae. The inhibition of the mitochondrial apoptosis pathway was also involved. Further research is required to determine the full underlying mechanisms by which Sal has an effect.

Platelet-activating factor (PAF), protein kinase C (PKC)βI, transforming growth factor (TGF)‑β1 and aberrant extracellular matrix (ECM) deposition have been associated with diabetic nephropathy (DN). However, the mechanistic basis underlying this association remains to be elucidated. The present study investigated the association among the aforementioned factors in a DN model consisting of human mesangial cells (HMCs) exposed to high glucose (HG) and lysophosphatidylcholine (LPC) treatments. HMCs were divided into the following treatment groups: Control; PAF; PAF+PKCβI inhibitor LY333531; HG + LPC; PAF + HG + LPC; and PAF + HG + LPC + LY333531. Cells were cultured for 24 h, and PKCβI and TGF‑β1 expression was determined using the reverse transcription‑quantitative polymerase chain reaction and western blotting. The expression levels of the ECM‑associated molecules collagen IV and fibronectin in the supernatant were detected using ELISA analysis. Subcellular localization of PKCβI was assessed using immunocytochemistry. PKCβI and TGF‑β1 expression was increased in the PAF + HG + LPC group compared with the other groups (P

Clara cell protein (CC16) is an anti-inflammatory protein, which is expressed in the airway epithelium. It is involved in the development of airway inflammatory diseases, including chronic obstructive pulmonary disease and asthma. However, the exact molecular mechanism underlying its anti‑inflammatory action remains to be fully elucidated. The aim of the present study was to define the protein profiles of the anti‑inflammatory effect of CC16 in lipopolysaccharide (LPS)‑treated rat tracheal epithelial (RTE) cells using shotgun proteomics. Protein extracts were obtained from control RTE cells, RTE cells treated with LPS and RTE cells treated with LPS and recombinant CC16 (rCC16). Subsequent label‑free quantification and bioinformatics analyses identified 12 proteins that were differentially expressed in the three treatment groups as a cluster of five distinct groups according to their molecular functions. Five of the twelve proteins were revealed to be associated with the cytoskeleton: Matrix metalloproteinase‑9, myosin heavy chain 10, actin‑related protein‑3 homolog, elongation factor 1‑α‑1 (EF‑1‑α‑1), and acidic ribosomal phosphoprotein P0. Five of the twelve proteins were associated with cellular proliferation: DNA‑dependent protein kinase catalytic subunit, EF‑1‑α‑1, tyrosine 3‑monooxygenase, caspase recruitment domain (CARD) protein 12 and adenosylhomocysteinase (SAHH) 3. Three proteins were associated with gene regulation: EF‑1‑α‑1, SAHH 3 and acidic ribosomal phosphoprotein P0. Three proteins were associated with inflammation: Tyrosine 3‑monooxygenase, CARD protein 12 and statin‑related protein. ATPase (H+‑transporting, V1 subunit A, isoform 1) was revealed to be associated with energy metabolism, and uridine diphosphate glycosyltransferase 1 family polypeptide A8 with drug metabolism and detoxification. The identified proteins were further validated using reverse transcription‑quantitative polymerase chain reaction. These protein profiles, and their interacting protein network, may facilitate the elucidation of the molecular mechanisms underlying the anti‑inflammatory effects of CC16.

Hepatitis B virus X protein (HBx) has been termed a viral oncoprotein, and is involved in the initiation and progression of hepatocellular carcinoma (HCC). Cyclooxygenase‑2 (COX‑2) and β‑catenin have been attributed to the oncogenic activity of HBx in HBV‑associated HCC. The present study aimed to determine whether there is crosstalk between COX‑2 and the Wnt/β‑catenin signaling pathway during HL‑7702‑HBx cell proliferation, and to investigate the associated underlying molecular mechanism. In the present study, cell proliferation assay, colony formation assay and flow cytometric analysis were used to detect the proliferative ability of cells. Reverse transcription‑quantitative polymerase chain reaction and western blotting were performed to examine the mRNA and protein expression of COX‑2, β‑catenin, cyclin‑D1 and c‑myc. The results demonstrated that HL‑7702‑HBx exhibited increased cell proliferation, higher colony formation efficiency and a shortened G1 period of the cell cycle. In addition, the mRNA and protein expression levels of COX‑2 were increased, and this was associated with HL‑7702‑HBx cell growth. Furthermore, the expression of β‑catenin and its target genes, cyclin‑D1 and c‑myc proto‑oncogene protein, was upregulated by HBx via COX‑2. Finally, HBx promoted HL‑7702 cell proliferation through the Wnt/β‑catenin signaling pathway. In conclusion, the primary finding of the present study was that HBx may promote HL‑7702 cell proliferation via the COX‑2/Wnt/β‑catenin pathway. Thus, it may be helpful to further investigate the molecular mechanism of HBV‑associated hepatocellular carcinoma.

Flavonol (−)-epicatechin (EPI) is primarily contained in green tea (Camellia sinensis) and cocoa beans (Theobroma cacao), and has been demonstrated to be beneficial for the health of the cardiovascular system. However, the effect and the underlying mechanism of EPI on myocardial ischemia induced cardiac injury has not yet been determined. Therefore, the present study aimed to detect whether EPI inhibited myocardial ischemia injury. An in vivo mouse myocardial ischemia model was induced by the ligation of left descending coronary artery for 7 days. EPI (1 mg/kg/day) was administrated 10 days prior to myocardial ischemia operation. The in vitro mouse myocardial ischemia model was induced by cultivating neonatal mouse cardiomyocytes under anoxia condition for 12 h. Cardiomyocytes were treated with EPI (5 µM) for 1 h and then incubated under anoxia conditions. Mouse hearts and cultured cardiomyocytes were used for hematoxylin-eosin, masson, ultrasonography, terminal dUTP nick end-labeling, immunofluorescence, western blotting and MTT assays. Results revealed that myocardial ischemia-induced mouse cardiac injury was significantly inhibited by EPI, as evidenced by decreased myocardial apoptosis, cardiac fibrosis and myocardial hypertrophy and improved cardiac function. In addition, it was confirmed that EPI serves a protective effect against myocardial ischemia via the phosphatase and tensin homolog (PTEN)/phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway, which was reversed by the PI3K inhibitor, {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002. The protective role of EPI in myocardial apoptosis was further confirmed on mouse cardiomyocytes following anoxia treatment in vitro. In conclusion, the data suggested that EPI protects against myocardial ischemia induced cardiac injury through the PTEN/PI3K/AKT signaling pathway in vivo and in vitro, which may provide clinical therapeutic approaches and targets for cardiac ischemia injury.

Serum exosomal microRNAs (miRNAs) have received considerable attention as potential biomarkers for tumor diagnosis. Reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) is commonly used to detect miRNA expression levels in various types of cancer. One prerequisite for valid RT‑qPCR data is the correct normalization of miRNAs to stably expressed endogenous reference genes (RGs). The study of liver carcinoma resection requires the use of reliable RGs in order to assess the expression levels of serum exosomal target miRNAs. However, the assessment of RG suitability for optimum serum exosomal miRNA expression analysis has yet to be investigated. The present study investigated the expression stability of 10 candidate RGs. The candidate genes included eight miRNAs (miR‑16, miR‑103, miR‑191, let‑7a, miR‑26a, miR‑221, miR‑181a, and miR‑451) and two small RNAs (5S and U6). The stability values of the candidate genes were calculated using the following algorithms: geNorm, NormFinder, BestKeeper, and the comparative ΔCt method. The overall ranking obtained from these analyses revealed that miR‑221, let‑7a, and miR‑26a were appropriate internal RGs for analysis of serum miRNAs in patients with hepatocellular carcinoma. In addition, normalization with miR‑221 and let‑7a combined, as recommended by geNorm, or with miR‑26a, as recommended by NormFinder, increased the accuracy of interpretation of the target miRNA expression levels in hepatopathy studies.

Denervated-dependent skeletal muscle atrophy (DSMA) is a disorder caused by the peripheral neuro-disconnection of skeletal muscle. The current study aimed to investigate the molecular mechanism and potential therapeutic strategies for the DSMA. A DSMA rat model was established. A lentiviral vector expressing small interfering RNA (siRNA) targeting angiopoietin-like protein 4 (ANGPTL4) was generated and injected into the rats that were also treated with Buyang Huanwu Tang (BYHWT). Reverse transcription-quantitative polymerase chain reaction was performed to examine ANGPTL4 mRNA expression in anterior cervical muscle samples. Western blot assay was used to evaluate ANGPTL4, nuclear factor-κB (NF-κB) and muscle RING-finger protein-1 (MURF1) expression. The ultrastructure of muscle tissues was viewed using transmission electron microscopy. The cell apoptosis in muscle tissues was detected using the terminal deoxynucleotidyl transferase dUTP nick end labeling. The results indicated that BYHWT treatment increased ANGPTL4 mRNA and protein levels in muscle tissues. The suppression of ANGPTL4 using siRNA significantly increased inflammatory cells compared with the control siRNA group. BYHWT protected the ultrastructure muscle tissues and inhibited cell apoptosis in the DSMA model. The protective effect of BYHWT protected may be mediated by increased expression of NF-κB p65 and MURF1. In conclusion, BYHWT may improve denervation-dependent muscle atrophy by increasing ANGPTL4 expression, involving NF-κB and MURF1 signaling.

Acute renal failure is one of the most common complications observed in hospitals. Although extensive studies have been carried out to search for therapeutic treatments, no effective cure has been established. In recent years, stem cell therapy for tissue engineering and repair has become a key area of study. Bone marrow mesenchymal stem cells (MSCs) have been demonstrated to exhibit a reparative role in ischemia reperfusion-injured kidney tissue, and the stromal cell-derived factor-1 (SDF-1)/chemokine (C-X-C motif) receptor 4 (CXCR4) axis has been found to play an important role in the migration and homing of MSCs. In the present study, transforming growth factor-β1 (TGF-β1) in the homogenate of the acute ischemia reperfusion-injured renal tissue was found to markedly increase the CXCR4 surface expression of MSCs, which contributes to the migration of MSCs to SDF-1. Neutralization of TGF-β1 inhibited the migration in an antibody concentration-dependent manner, through downregulation of CXCR4 localized to the membrane. These observations suggest a potential mechanism for MSC migration to the kidney which may provide a possible therapeutic target for curing acute renal failure.

An eight‑base pair (bp) deletion in the Pou4f3 gene in hair cells is associated with DFNA15, a hereditary form of hearing loss. To explore the pathological mechanisms underlying the development of DFNA15, the effect of the mutation in Pou4f3 on the activity of the myosin VI (Myo6) promoter, was investigated. The upstream regulatory sequence of Myo6 (2625 bp), consisting of an 1899 bp upstream sequence and a 727 bp intron 1 sequence, was amplified using polymerase chain reaction and subcloned into the pGL3‑Basic vector expressing firefly luciferase. For verification of inserted fragments, plasmids were subjected to restriction analysis and then sequenced. HEK293T human embryonic kidney cells were transiently transfected with renilla luciferase‑thymidine kinase vectors expressing Renilla luciferase and the Myo6 promoter‑driven firefly luciferase expressing vectors along with pIRES2‑enhanced green fluorescent protein (EGFP)‑Pou4f3 (expressing wild‑type Pou4f3) or pIRES2‑EGFP‑Pou4f3 (expressing the truncation mutant of Pou4f3). The relative luciferase activities were measured to determine the activity of the Myo6 promoter. The Myo6 promoter activity was not affected by co‑expression of wild‑type Pou4f3, as indicated by the comparable relative luciferase activities in the presence of the pIRES2‑EGFP‑Pou4f3 and the empty control vectors. However, co‑expression of mutated Pou4f3 significantly inhibited the activity of the Myo6 promoter to almost half of that of the control (P

General anesthesia is widely used in pediatric surgery, although the influence of general anesthesia on cerebellar information transmission and motor function is unclear. In the present study, neonatal mice received repeated inhalation of sevoflurane, and electrophysiological alterations in Purkinje cells (PCs) and the development of motor functions were detected. In addition, γ-aminobutyric acidA receptor e (GABAA-R e) subunit knockout mice were used to investigate the mechanism of action of sevoflurane on cerebellar function. In the neonatal mice, the field potential response of PCs induced by sensory stimulation and the motor function indices were markedly inhibited by sevoflurane, and the inhibitory effect was positively associated with the number of repetitions of anesthesia. In additional the GABAA-R e subunit level of PCs was promoted by sevoflurane in a dose-dependent manner, and the inhibitory effects of sevoflurane on PC field potential response and motor function were alleviated in GABAA-R e subunit knockout mice. The GABAA-R e subunit was activated by sevoflurane, leading to inhibition of sensory information transmission in the cerebellar cortex, field potential responses of PCs and the development of cerebellar motor function. The present study provided experimental evidence for the safe usage of sevoflurane in clinical anesthesia, and suggested that GABAA-R e subunit antagonists may be considered for combined application with general anesthesia with repeated inhalation of sevoflurane, for adverse effect prevention in the clinic.

Intracerebral hemorrhage (ICH) results in inflammation, and glucocorticoids have been proven to be effective inhibitors of ICH‑induced inflammation. However, the precise underlying mechanisms of ICH‑induced inflammation and glucocorticoid function remain largely undefined. Using a mouse ICH model, the present study demonstrated that the short non‑coding RNA molecule microRNA‑155 (miR‑155) is involved in the inflammatory process initiated by ICH in mice. Increased mRNA expression levels of miR‑155, as well as the pro‑inflammatory cytokines interferon‑β (IFN‑β), tumor necrosis factor‑α (TNF‑α) and interleukin‑6 (IL‑6), were observed in vivo following ICH. By contrast, the expression level of suppressor of cytokine signaling 1 (SOCS‑1) protein was reduced in the ICH group compared with control mice. Similar results were observed in vitro using astrocytes, the primary effector cells in ICH. Compared with wild type astrocytes, astrocytes overexpressing miR‑155 exhibited significant inhibition of SOCS‑1 protein expression levels. These results suggest that miR‑155 contributes to the development of ICH‑induced inflammation in mice by downregulating SOCS‑1 protein expression levels and promoting pro‑inflammatory cytokine (IFN‑β, TNF‑α and IL‑6) production. Expression levels of miR‑155 and pro‑inflammatory cytokines in the ICH group were significantly decreased following dexamethasone administration. This suggests that glucocorticoids attenuate ICH‑induced inflammation by targeting the miR‑155/SOCS‑1 signaling pathway in mice. In conclusion, the results of the present study demonstrated that the miR‑155/SOCS‑1 signaling pathway is required for ICH‑induced inflammation, and glucocorticoids inhibit this process by targeting the miR‑155/SOCS‑1 signaling pathway.

The purpose of this study was to analyze the effects of bufalin on the gene expression of K562 cells and on the expression of BMI‑1 pathway constituents in K562 cell apoptosis. K562 cells were treated with bufalin, and the inhibition rate and apoptosis were detected by an MTT assay, flow cytometry and a microarray assay. BMI‑1, p16INK4a and p14ARF were examined by quantitative polymerase chain reaction (qPCR). Bufalin induced significant changes in the gene expression of the K562 cells; 4296 genes were differentially expressed, 2185 were upregulated and 2111 were downregulated. The most upregulated genes were associated with transcription regulation, while the most downregulated genes were associated with the non-coding RNA metabolic processes and DNA repair. qPCR analysis demonstrated that BMI‑1 was overexpressed in the K562 cells. Bufalin is able to downregulate BMI‑1 expression levels in K562 cells prematurely and cause an increase in the expression levels of p16INK4a and p14ARF. Moreover, bufalin downregulated BCR/ABL expression levels in a time‑dependent manner, and the expression of BCR/ABL was not associated with the upregulation or downregulation of BMI‑1 expression. Bufalin may induce K562 cell apoptosis by downregulating BMI‑1 expression levels and accordingly upregulating the expression levels of p16INK4a and p14ARF. Bufalin may also induce K562 cell apoptosis via downregulating BCR/ABL expression levels, and this pathway may be independent of the BMI‑1 pathway.