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Renal cell carcinoma has the highest incidence rate of cancer types in the urinary system. Moreover, microRNAs (miRNA) have been closely associated with numerous types of tumor. The present study aimed to investigate the effects of miRNA (miR)-133b on the proliferation, invasion and chemosensitivity of renal cell carcinoma cells, and to determine whether its mechanism was regulated by the ERK signaling pathway. Both renal cell carcinoma and adjacent healthy tissues from 60 patients, in addition to renal cell carcinoma lines, ACHN, Caki-1, A-498 and 786-O, and 293 cells, were used in this study. miR-133b expression was measured from renal cell carcinoma, adjacent healthy tissues and renal cell carcinoma cell lines by reverse transcription-quantitative PCR. Cells were transfected with miR-133b mimic to achieve miR-133b overexpression. The proliferative, migratory and invasive ability of the cells were evaluated using MTT, wound healing and Matrigel assays, respectively, and flow cytometry was used to detect the apoptotic rate. Following treatment with an ERK inhibitor, U0126, and activator, LM22B-10, western blotting was used to detect the expression of related proteins and the activity of the ERK signaling pathway. The overexpression of miR-133b significantly inhibited cell proliferation, migration and invasion, whilst inducing apoptosis and increasing the drug sensitivity of renal cell carcinoma cells to cisplatin, docetaxel and doxorubicin. The miR-133b mimic also increased the protein expression levels of Bax and decreased the expression levels of matrix metalloproteinase (MMP)-2, MMP-9, ATP-binding cassette subfamily G2, P-glycoprotein, Bcl-2 and proliferating cell nuclear antigen, as well as the phosphorylation of ERK (P0.05). However, the overexpression of miR-133b combined with LM22B-10 treatment weakened the anticancer effects of miR-133b mimic transfection (P

MicroRNA (miR)-19b is expressed in various types of tumors and may serve as a potential therapeutic target. The miR‑17‑92 cluster is upregulated in nasopharyngeal carcinoma (NPC) tissues and cells. miR‑19b is a member of the miR‑17‑92 cluster; however, its expression and function in NPC are largely unknown. The present study aimed to investigate the expression and function of miR‑19b in NPC cells. The miRCURY LNATM miRNA Inhibitor (miR‑19b inhibitor and negative control) were transfected into C666‑1 cells. The proliferation, apoptosis and migration of the cells were subsequently detected by the Cell Counting Kit‑8 assay, flow cytometry and Transwell assay, respectively. Additionally, the expression of STAT3 signaling pathway‑associated proteins [STAT3, pSTAT3 and suppressor of cytokine signaling 1 (SOCS1)] and the transcriptional targets of pSTAT3 [Bcl‑2, myeloid leukemia protein 1 (Mcl‑1) and cyclin D1] were detected by western blotting. The miR‑19b inhibitor inhibited proliferation and migration and induced apoptosis of C666‑1 cells. Furthermore, the miR‑19b inhibitor upregulated the expression of SOCS1, a predicted target gene of miR‑19b, and decreased the phosphorylation of STAT3 at Tyr705 and Ser727. These data indicated that upregulation of SOCS1, an endogenous inhibitor of STAT3 phosphorylation, attenuated the STAT3 signaling pathway in C666‑1 cells. Moreover, the expression level of the proproliferative protein cyclin D1 and antiapoptotic proteins Mcl‑1 and Bcl‑2 was significantly decreased following transfection with the miR‑19b inhibitor. The aforementioned three proteins are downstream transcriptional targets of the activated STAT3 signaling pathway. The results of the present study revealed that inhibition of miR‑19b negatively modulated the malignant behavior of NPC cells via the STAT3 signaling pathway. Therefore, miR‑19b inhibition may serve as a novel therapeutic target for the treatment of NPC.

Modulating the biological status of endothelial progenitor cells (EPCs), such as function and survival, is essential for therapeutic angiogenesis in ischemic vascular disease environments. This study aimed to explore the role and molecular mechanisms underlying Netrin‑1 in the viability and angiogenic function of EPCs. EPCs were isolated from the bone barrow of adult C57/BL6 mice. The apoptosis and various functions of EPCs were analyzed in vitro by manipulating the expression of Netrin‑1. The TUNEL assay was performed to detect apoptotic EPCs. Cell migration and tube formation assays were performed to detect EPC function. Trypan blue staining was performed to detect cell viability. Western blot analysis was performed to detect the protein expression levels of Netrin‑1, CD146 and apoptotic factors. Quantitative PCR analysis was performed to detect the expression levels of Netrin‑1 receptors. The results demonstrated that treatment with exogenous Netrin‑1 promoted EPC migration and tube formation, whereas transfection with small interfering (si)RNA targeting Netrin‑1 exhibited the opposite effects. Exogenous Netrin‑1 protected EPCs from hypoxia‑induced apoptosis, whereas the interruption of endogenous Netrin‑1 enhancement under hypoxia by Netrin‑1‑siRNA exacerbated the apoptosis of EPCs. Furthermore, CD146, one of the immunoglobulin receptors activated by Netrin‑1, was screened for in the present study. Results demonstrated that CD146 did not participate in Netrin‑1‑promoted EPC function, but mediated the anti‑apoptotic effects of Netrin‑1 in EPCs. In conclusion, Netrin‑1 enhanced the angiogenic function of EPCs and alleviated hypoxia‑induced apoptosis, which was mediated by CD146. This biological function of Netrin‑1 may provide a potential therapeutic option to promote EPCs for the treatment of ischemic vascular diseases.

Gastric cancer (GC) is a leading cause of cancer‑associated mortality worldwide. Previous studies demonstrated that long noncoding RNAs (lncRNAs) may be dysregulated in GC and may serve important roles in cancer progression. The present study aimed to investigate the role of the novel lncRNA stomach cancer‑associated transcript 16 (STCAT16; Assembly Gene ID G038291) in the development and progression of GC. The present data suggested that the expression level of STCAT16 was decreased in GC tissues. The expression level of STCAT16 was identified to be associated with lymph node and tumour node metastasis stages. Furthermore, the expression level of STCAT16 was identified to be significantly associated with poor survival and prognosis. Knockdown of STCAT16 promoted proliferation, colony formation, migration and invasion of BGC‑823 cells. In contrast, these features were suppressed in AGS cells following overexpression of STCAT16. In vivo, tumour growth was significantly decreased following STCAT16 overexpression. Collectively, the present data suggested that the lncRNA STCAT16 may act as a tumour suppressor and may inhibit GC tumour cell growth and migration. Additionally, the decreased expression level of STCAT16 was identified to be associated with poor prognosis in patients with GC.

Atherosclerosis (AS) is a progressive disease of multifactorial origin, which occurs in response to endothelial injury. Increased homocysteine (Hcy) is considered a major cause of endothelial dysfunction, oxidative stress and DNA methylation; however, the mechanisms remain to be fully elucidated. The aim of the present study was to investigate whether Hcy causes injury to endothelial cells (ECs) by the effect of lectin‑like oxidized‑low density lipoprotein receptor‑1 (LOX‑1) DNA methylation through toll‑like receptor 4(TLR4)/nuclear factor (NF)‑κB/DNA methyltransferase (DNMT)1. The ECs were treated with different concentrations of Hcy, and it was found that Hcy promoted the expression of TLR4, leading to EC injury. The effect of oxidative stress was analyzed by measuring superoxide dismutase, malondialdehyde and hydrogen peroxide in the ECs. In addition, the association between NF‑κB and DNMT1 was examined by treatment of the ECs with pyrrolidine dithiocarbamate (PDTC). The results suggested that Hcy induced LOX‑1 DNA hypomethyaltion to promote the expression levels of LOX‑1. Taken together, Hcy injured the ECs through the effect of methylation and trans‑sulfuration metabolism of LOX‑1 through TLR4/NF‑κB/DNMT1. Following injury to the ECs, lipids, particularly ox‑LDL, accumulated in the sub‑endothelial layer to promote the formation of AS.

Multidrug resistance (MDR) is a major clinical obstacle in the successful treatment of patients with metastatic nasopharyngeal carcinoma (NPC). Results from previous studies suggest that microRNAs (miRNA) may be involved in promoting MDR in multiple cancer types. However, the role of miR‑125b in modulating the MDR of NPC is elusive. In the present study, miR‑125b expression in cisplatin (DDP) ‑resistant CNE2 cells (CNE2/DDP) was compared with parental counterparts, using reverse transcription‑quantitative polymerase chain reaction. A >3‑fold reduction in miR‑125b expression levels was observed in CNE2/DDP cells compared with parental CNE2 cells. Ectopic expression of miR‑125b by transfecting CNE2/DDP cells with miR-125b mimics, increased DDP‑induced cytotoxicity, apoptosis and chemosensitivity. By contrast, suppression of miR-125b by transfecting CNE2 cells with miR‑125b inhibitors, reduced DDP‑induced cytotoxicity and apoptosis, and facilitated cisplatin resistance. The results suggest that miR‑125b may regulate the sensitivity of NPC cells to DDP by modulating the expression levels of antiapoptotic factor B-cell CLL/lymphoma 2. Collectively, the results of the present study highlight miR‑125b as a potential therapeutic target for reversing MDR in NPC.

Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease characterized by chronic inflammation, fibroblast proliferation and extracellular matrix deposition. However, the molecular and cellular mechanisms underlying the pathogenesis of pulmonary fibrosis remain to be fully elucidated. The contribution of the phosphoinositide 3‑kinase (PI3K)/protein kinase B (Akt) pathway in fibrotic processes remains to be investigated. The aim of the present study was to investigate the role of the PI3K/Akt pathway in pulmonary fibrosis. A rat model of pulmonary fibrosis was induced by intratracheal administration of bleomycin (BLM), and a specific PI3K/Akt inhibitor, LY294002, was used to assess the role of the PI3K/Akt pathway in fibrogenesis. The inflammatory and fibrotic alterations in the lung tissues were evaluated using histological staining and the hydroxyproline assay. In addition, the concentration of cytokines in bronchoalveolar lavage fluid and the expression of Akt, phosphorylated (p‑)Akt, epithelial cadherin, α smooth muscle actin and vimentin in lung tissues. The data demonstrated that an increase in the expression levels of p‑Akt was involved in the progression of pulmonary fibrosis and contributed to fibrogenesis. Administration of the Akt inhibitor significantly attenuated inflammation and fibrosis, which was accompanied by a reversal of lung fibrosis‑associated epithelial‑mesenchymal transition. Taken together, these observations suggest that the PI3K/Akt pathway serves a central role in the pathophysiology of lung fibrosis, and is a promising therapeutic target.

Sodium taurocholate cotransporting polypeptide (NTCP), a carrier protein encoded by solute carrier family 10 member 1 (SLC10A1), is expressed in the basolateral membrane of hepatocytes, where it is responsible for the uptake of bile acids from plasma into hepatocytes. The first patient with NTCP deficiency was described in 2015. A limited number of such patients have been reported in the literature and their genotypic and phenotypic features require further investigation. The current study investigated 4 patients with NTCP deficiency from two unrelated families. The patients were subjected to SLC10A1 genetic analysis and it was revealed that all patients were compound heterozygous for the c.800C>T (p.Ser267Phe) and c.595A>C (p.Ser199Arg) SLC10A1 variants. To the best of the authors' knowledge, the latter variant had not been previously reported. Further analysis in 50 healthy individuals did not identify carriers. The c.595A>C (p.Ser199Arg) variant exhibited co-segregation with hypercholanemia and exhibited a relatively conserved amino acid when compared with homologous peptides. Moreover, SWISS-MODEL prediction revealed that the mutation affected the conformation of the NTCP molecule. The 4 patients demonstrated varying degrees of hypercholanemia while a downward trend in the plasma levels of total bile acids (TBA) in 2 pediatric patients and occasionally normal TBA level in an adult case were observed. The results indicated an autosomal recessive trait for NTCP deficiency, supported the primary role of NTCP in the uptake of bile acids from plasma and suggested that hepatic uptake of bile acids may occur by means other than NTCP uptake. Moreover, the novel missense variant c.595A>C(p.Ser199Arg) enriched the SLC10A1 mutation spectrum and may serve as a new genetic marker for the molecular diagnosis and genetic counseling of NTCP deficiency.

Alzheimer's disease (AD) is a chronic neurodegenerative, and abnormal aggregation of the neurotoxic β amyloid (Aβ) peptide is an early event in AD. The present study aimed to determine the correlation between the nicotinic acetylcholine receptor α7 subunit (α7 nAChR) and Aβ in the brains of patients with AD, and to investigate whether the increased expression levels of the α7 nAChR could alter the neurotoxicity of Aβ. The expression levels of α7 nAChR and Aβ in the brains of patients with AD and healthy brains were analyzed using immunofluorescence. Moreover, SH‑SY5Y cells were used to stably overexpress or silence α7 nAChR expression levels, prior to the treatment with or without 1 µmol/l Aβ1‑42 oligomer (AβO). The mRNA and protein expression levels of α7 nAChR, synaptophysin (SYP), postsynaptic density of 95 kDa (PSD‑95) and synaptosomal‑associated protein of 25 kDa (SNAP‑25) were subsequently analyzed using reverse transcription‑quantitative PCR and western blotting. In addition, the concentration of acetylcholine (ACh) and the activity of acetylcholinesterase (AChE) were analyzed using spectrophotometry, while the cell apoptotic rate was determined using flow cytometry. The expression of Aβ in the brains of patients with AD was found to be significantly increased, whereas the expression of α7 nAChR was significantly decreased compared with the healthy control group. In vitro, the expression levels of α7 nAChR were significantly increased or decreased following the overexpression or silencing of the gene, respectively. Consistent with these observations, the mRNA and protein expression levels of SYP, PSD‑95 and SNAP‑25 were also significantly increased following the overexpression of α7 nAChR and decreased following the genetic silencing of the receptor. In untransfected or negative control cells, the expression levels of these factors and the apoptotic rate were significantly reduced following the exposure to AβO, which was found to be attenuated by α7 nAChR overexpression, but potentiated by α7 nAChR RNA silencing. However, no significant differences were observed in either the ACh concentration or AChE activity following transfection. Collectively, these findings suggested that α7 nAChR may protect the brains of patients with AD against Aβ, as α7 nAChR overexpression increased the expression levels of SYP, SNAP‑25 and PSD‑95, and attenuated the inhibitory effect of Aβ on the expression of these synaptic proteins and cell apoptosis. Overall, this indicated that α7 nAChR may serve an important neuroprotective role in AD.

17β‑estradiol (E2) and aquaporin 2 (AQP2) are associated with endometrial receptivity, and E2 directly regulates AQP2 expression in endometrial cancer cells. The present study aimed to investigate the role of AQP2 in embryo implantation. Normal endometrial samples were collected at the Women's Hospital (Hangzhou, China) from women seeking in vitro fertilization and embryo transfer; women with endometrial abnormalities were excluded from the study. Samples were categorized into early‑mid proliferative, late proliferative, early secretory, mid‑secretory and late secretory phase groups, according to the menstrual cycle. The mRNA and protein expression levels of AQP2 were assessed in normal human endometrium in response to E2 via reverse transcription‑quantitative polymerase chain reaction and western blotting, respectively. The effects of AQP2 on spheroid attachment were assessed using an in vitro co‑culture assay with small interfering (si)RNA against AQP2. The highest expression levels of AQP2 were observed in the late proliferative and mid‑secretory phases, with the lowest levels detected in the early proliferative and late secretory phases. In addition, treatment with 10‑9 or 10‑7 M E2 for 24 h upregulated AQP2 in the cultured endometrium. Knockdown of AQP2 by siRNA significantly decreased JAr spheroid attachment; however, this effect was significantly reversed when AQP2 siRNA‑transfected cells were treated with 10‑7 M E2. The results of the present study suggested that AQP2 expression levels in human endometrium may be mediated by estrogen, and low AQP2 expression levels may be a potential cause of impaired uterine receptivity.

The purpose of the present study was to explore aging-associated cardiac dysfunction and the possible mechanism by which swimming exercise modulates cardiac dysfunction in aged mice. Aged mice were divided into two groups: i) Aged mice; and ii) aged mice subjected to swimming exercises. Another cohort of 4-month-old male mice served as the control group. Cardiac structure and function in mice were analyzed using hematoxylin and eosin staining, and echocardiography. The levels of oxidative stress were determined by measuring the levels of superoxide dismutase, malondialdehyde and reactive oxygen species (ROS). Levels of the endoplasmic reticulum (ER) stress-related protein PKR-like ER kinase, glucose-regulated protein 78 and C/EBP homologous protein were determined to evaluate the level of ER stress. The aged group exhibited an abnormal cardiac structure and decreased cardiac function, both of which were ameliorated by swimming exercise. The hearts of the aged mice exhibited pronounced oxidative and ER stress, which were ameliorated by exercise, and was accompanied by the reactivation of myocardial cGMP and suppression of cGMP-specific phosphodiesterase type 5 (PDE5). The inhibition of PDE5 attenuated age-induced cardiac dysfunction, blocked ROS production and suppressed ER stress. An ER stress inducer abolished the beneficial effects of the swimming exercise on cardiac function and increased ROS production. The present study suggested that exercise restored cardiac function in mice with age-induced cardiac dysfunction by inhibiting oxidative stress and ER stress, and increasing cGMP-protein kinase G signaling.

The aim of the present study was to identify an effective method for detecting early‑phase atherosclerosis (AS), as well as to provide useful DNA methylation profiles to serve as biomarkers for the detection of AS. A total of 300 individuals (150 AS patients and 150 healthy subjects) were recruited for peripheral blood DNA methylation analyses at 12 gene promoter loci using nested methylation‑specific polymerase chain reaction in a test set. Based on the test set, the promoter methylation of TIMP metallopeptidase inhibitor 1 (TIMP1), ATP binding cassette subfamily A member 1 (ABCA1), and acetyl-CoA acetyltransferase 1 (ACAT1) were determined to be candidate biomarkers; demonstrating the highest sensitivity (88%) and specificity (90%). The biomarkers that were candidates for early AS detection were validated in an independent validation set (n=100). In the validation set, the combination of TIMP1, ABCA1 and ACAT1 methylation achieved sensitivity, specificity and coincidence rate values of 88, 70 and 79%, respectively. In the current pilot study, the patterns of DNA methylation of AS‑associated genes were observed to be significantly altered in the peripheral blood of AS patients. Thus, the AS-specific methylation of the three‑gene panel (TIMP1, ABCA1, and ACAT1) may serve as a valuable biomarker for the early detection of AS.

Mitochondrial dysfunction has been increasingly associated with the development of cardiovascular diseases, including hypertension and cardiac hypertrophy. In the present study, NADH dehydrogenase 1α subcomplex 10 (Ndufa10) was characterized from the left ventricular muscles of spontaneously hypertensive rats (SHRs) and normal Wistar Kyoto (WKY) rats. Western blot analysis demonstrated that there was a shift in the molecular weight (MW) and in the isoelectric point (pI) of the Ndufa10 protein from SHRs and WKY rats. Mass spectrometric analysis revealed that the replacement of an aspartate residue with asparagine at amino acid position 120 was the biochemical difference between the two Ndufa10 isoforms. Further analysis using the bacterially expressed proteins Ndufa10‑120N (WKY) and Ndufa10‑120D (SHR) revealed that the shift in the pI and MW of the two Ndufa10 isoforms was solely caused by the amino acid mutation, and not by post‑translational modifications. Since deficiencies of the mitochondrial complex I are the most common defects in the oxidative phosphorylation system, further studies are required to study the difference between the activities of the two Ndufa10 variants, and their role in the pathogenesis of hypertension.

Accumulating evidence has suggested that homocysteine (Hcy) is an independent risk factor for atherosclerosis (AS). Hcy can promote vascular smooth muscle cell (VSMC) proliferation, which is pivotal in the pathogenesis and progression of AS. The aim of the present study was to investigate the epigenetic regulatory mechanism of microRNA (miR)‑143‑mediated VSMCs proliferation induced by Hcy. The results of a 3‑(4,5‑dimethylthiazol‑2‑yl)‑2,5‑diphe‑nyltetrazolium bromide assay revealed that VSMC proliferation was increased by 1.39‑fold following treatment with 100 mM Hcy, compared with the control group. The levels of miR‑143 were markedly downregulated in the Hcy group, compared with the control group, as determined using reverse transcription‑quantitative polymerase chain reaction analysis. In addition, the level of miR‑143 methylation was increased markedly in the VSMCs treated with Hcy, compared with the control, and was reduced following transfection with DNA methyltransferase (DNMT)3a small interfering RNA, determined using methylation‑specific‑PCR. The activities of DNMT3a luciferase were also altered accordingly in VSMCs transfected with pre‑miR‑143 and miR‑143 inhibitor, respectively. In addition, the expression of miR‑143 was observed to be inversely correlated with the mRNA and protein expression of DNMT3 in the VSMCs. Taken together, these findings suggest that DNMT3a is a direct target of miR‑143, and that the upregulation of DNMT3 is responsible for the hypermethylation of miR‑143 in Hcy-induced VSMC proliferation.

Long non‑coding RNAs (lncRNAs) are a specific group of RNA molecules that do not encode proteins. They have been shown to serve important regulatory functions in various biological and cell differentiation processes. However, the potential functions and regulatory mechanisms of lncRNAs that are associated with the osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs) remain to be elucidated. The present study aimed to investigate lncRNAs that are differentially expressed during the osteogenic differentiation of hBMSCs, along with the potential functions of those lncRNAs. To this end, three groups of hBMSCs were stimulated to undergo osteogenic differentiation for 7 days. Known lncRNAs, unknown lncRNAs and mRNAs that demonstrated differential expression prior to and following the osteogenic differentiation of hBMSCs were screened using lncRNA high‑throughput sequencing. In addition, 12 lncRNAs were selected for reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) validation of the accuracy of the sequencing results. The potential functions and possible targets of the differentially expressed lncRNAs were analyzed using bioinformatics technologies (gene ontology, Kyoto Encyclopedia of Genes and Genomes and gene co‑expression network analysis). In total, 64 lncRNAs were differentially expressed by at least two‑fold in hBMSCs prior to and following osteogenic differentiation; these included seven known lncRNAs (two upregulated and five downregulated lncRNAs) and 57 unknown lncRNAs (35 upregulated and 22 downregulated lncRNAs). In addition, 409 mRNAs (257 upregulated and 152 downregulated mRNAs) were differentially expressed by at least two‑fold. The RT‑qPCR results obtained for 12 selected differentially expressed lncRNAs were consistent with the sequencing results. The gene co‑expression network analysis of lncRNAs and mRNAs demonstrated that four lncRNAs (ENSG00000238042, lnc_1269, lnc_1369 and lnc_1708) may serve important roles in the osteogenic differentiation of hBMSCs. In conclusion, during the osteogenic differentiation of hBMSCs, the lncRNA expression profile changed significantly; certain of the observed differentially expressed lncRNAs may be derived from protein‑coding genes and may serve important roles in osteogenic differentiation.

Nasopharyngeal carcinoma (NPC) is a highly invasive malignancy with cervical lymphopathy as the initial presentation. Epithelial-mesenchymal transition (EMT), a process by which epithelial cells lose cell-cell adhesion and gain migratory and invasive properties, has a pivotal role in metastasis. Forkhead box C1 (FoxC1), a member of the forkhead family of transcription factors, induces EMT and has a critical role in metastasis of multiple human cancers. However, the role of FoxC1 in the progression of NPC has remained elusive. The present study revealed that the expression of FoxC1 was markedly elevated in NPC tissues compared with that in chronically inflamed nasopharyngeal tissues and was closely correlated with vimentin, fibronectin and N-cadherin expression as indicated by immunohisto-chemical assays. In addition, high FoxC1 expression was positively associated with lymph node metastasis, distant metastasis and an advanced clinical stage in patients with NPC. Furthermore, FoxC1 expression was high in NPC cell lines while being low in an immortalized normal nasopharyngeal epithelial cell line. In vitro, knockdown of FoxC1 in the CNE2 human NPC cell line by small interfering RNA downregulated vimentin, fibronectin and N-cadherin expression and reduced the migratory and invasive capacity of CNE2 cells. In conclusion, the present study indicated that FoxC1 has a pivotal role in EMT through the upregulation of vimentin, fibronectin and N-cadherin expression. Thus, FoxC1 may be a potential therapeutic target in NPC.

Vascular smooth muscle cell (VSMC) proliferation is a primary pathological event in the development of atherosclerosis (AS), and the presence of homocysteine (Hcy) acts as an independent risk factor for AS. However, the underlying mechanisms remain to be elucidated. Phosphatase and tensin homologue on chromosome 10 (PTEN), is endogenously expressed in VSMCs and induces multiple signaling networks involved in cell proliferation, survival and inflammation, however, the specific role of PTEN is still unknown. The present study detected the proliferation ratio of VSMCs following treatment with Hcy and Resveratrol (RSV). In the 100 µM Hcy group, the proliferation ratio increased, and treatment with RSV decreased the proliferation ratio induced by Hcy. Reverse transcription‑quantitative polymerase chain reaction and western blotting were used to analyze PTEN expression, RSV treatment was associated with decreased PTEN expression levels in VSMCs. PTEN levels were decreased in Hcy treated cells, and the proliferation ratio of VSMCs were increased following treated with Hcy. To study the mechanism of regulation of PTEN by Hcy, the present study detected PTEN methylation levels in VSMCs, and PTEN DNA methylation levels were demonstrated to be increased in the 100 µM Hcy group, whereas treatment with RSV decreased the methylation status. DNA methyltransferase 1 is important role in the regulation of PTEN methylation. Overall, Hcy impacts the methylation status of PTEN, which is involved in cell proliferation, and induces the proliferation of VSMCs. This effect is alleviated by treatment with RSV, which exhibits an antagonistic mechanism against Hcy.

Vascular smooth muscle cell (VSMC) proliferation is a primary pathological event in atherosclerosis (AS), and homocysteine (Hcy) is an independent risk factor for AS. However, the underlying mechanisms are still lagging. Studies have used the combination of methylation of promoters of multiple genes to diagnose tumors, thus the aim of the current study was to investigate the role of methylation status of several genes in VSMCs treated with Hcy. CpG islands were identified in the promoters of platelet‑derived growth factor (PDGF), p53, phosphatase and tensin homologue on chromosome 10 (PTEN) and mitofusin 2 (MFN2). Hypomethylation was observed to occur in the promoter region of PDGF, hypermethylation in p53, PTEN and MFN2, and hypomethylation in two global methylation indicators, aluminium (Alu) and long interspersed nucleotide element‑1 (Line‑1). This was accompanied by an increase in the expression of PDGF, and reductions of p53, PTEN and MFN2, both in mRNA and protein levels. An elevation of S‑adenosylmethionine (SAM) and a reduction of S‑adenosylhomocysteine (SAH) and the SAM/SAH ratio were also identified. In conclusion, Hcy impacted methylation the of AS‑associated genes and global methylation status that mediate the cell proliferation, which may be a character of VSMCs treated with Hcy. The data provided evidence for mechanisms of VSMCs proliferation in AS induced by Hcy and may provide a new perspective for AS induced by Hcy.

The aim of the current study was to investigate the expression of chemokine CCL20 in ulcerative colitis (UC) patients and in dextran sulfate sodium (DSS)-induced experimental colitis in mice. The expression of the CCL20 protein in colonic mucosa was detected in 65 UC patients and in 30 normal patients. A total of 40 female BALB/c mice were divided into a control and model group; the latter was fed a 5% DSS solution ad libitum for 7 days to induce experimental colitis. The disease activity index (DAI) and histological score were assessed and the expression of CCL20 mRNA and protein was determined. CCL20 was expressed in the UC group. It was either weakly expressed or not expressed in the normal control group. Additionally, the expression of CCL20 in the UC group was significantly higher compared with that in the control groups (P

The aim of the present study was to investigate the effect of hepatitis B virus X protein (HBx) on the expression of DNA methyltransferase (DNMT)3A/3B and suppressors of cytokine signaling‑1 (SOCS‑1), as well as promoter CpG island methylation of the SOCS‑1 gene. Stable hepatocyte cell lines expressing the HBx gene (pcDNA‑X/QSG7701) or an empty gene (pcDNA3.0/QSG7701) were established. Reverse transcription quantitative polymerase chain reaction (PCR) was used to detect the mRNA expression levels of DNMT3A/3B and SOCS‑1. Immunohistochemistry was used to detect the protein expression of DNMT3A/3B. Methylation‑specific PCR (MSP) was used to detect the methylation status of the SOCS‑1 gene promoter. The mRNA and protein expression levels of DNMT3A/3B were significantly higher in the pcDNA‑X/QSG7701‑transfected cells, compared with those in the pcDNA3.0/QSG7701 or non‑transfected QSG7701 cells (P

Gastric cancer (GC) remains one of the leading causes of cancer-associated mortality. Inhibitor of apoptosis-stimulating protein of p53 (iASPP) is a member of the inhibitory apoptosis-stimulating protein p53 family. The overexpression of iASPP has been detected in several types of tumor in humans. However, the role of iASPP in GC remains to be elucidated. The objectives of the present study were to detect the expression of iASPP in GC and examine the potential role of iASPP in GC cell lines. Using reverse transcription-quantitative polymerase chain reaction and western blot analyses, it was identified that the expression of iASPP in GC tissues and GC cell lines was higher compared with that in adjacent normal tissues and in a normal gastric mucosa cell line (GES-1). To examine the role of iASPP in GC cells, the expression of iASPP was inhibited using a small interfering (si)RNA against iASPP and it was observed that iASPP expression was significantly downregulated. Using MTT assays, colony-formation assays and flow cytometry, it was identified that the inhibition of iASPP was able to significantly inhibit the proliferation and colony forming ability and promote apoptosis in GC cells. To examine the role of iASPP in GC cells in vivo, GC cells, which were infected with iASPP-siRNA or control-siRNA were subcutaneously injected into nude mice. It was identified that downregulation of iASPP significantly inhibited tumor growth in vivo. Thus, iASPP may be a potential molecular target in GC therapy.

Triptolide (TL) is known to suppress the proliferation of a number of pancreatic cancer cell lines in vitro. Marked antitumor effects were also observed in a xenograft model of pancreatic cancer. Hypoxia‑inducible factor‑1α (HIF‑1α) is highly expressed in pancreatic cancer cells lines. The present study therefore tested the hypothesis that suppression of HIF‑1α is associated with the antitumor activity of TL. Quantitative polymerase chain reaction and western blot analysis were used to determine the level of gene expression. A xenograft tumor model of pancreatic cancer was established in athymic nude mice and the tumor size was measured to evaluate the outcome of TL treatment. Immunohistochemistry was used to detect the expression of HIF‑1α and vascular endothelial growth factor (VEGF), and to assess microvessel density. Microarray was used to investigate gene expression in pancreatic cancer cells following TL treatment. The expression of HIF‑1α was shown to be reduced in pancreatic cell lines following treatment with TL, and this effect occurred in a dose‑dependent manner. In a xenograft model of pancreatic cancer, reduced levels of HIF‑1α were also observed in mice that were treated with TL. Furthermore, the expression of VEGF, which is a direct target of HIF‑1α, was also suppressed, and the microvessel density of tumor tissues was consequently reduced. A microarray analysis of gene expression was performed in order to investigate the potential mechanisms underlying the antitumor activity of TL. The results showed that 11 genes, including c‑Myc, SOX9 and Ets2, were downregulated at an early stage following treatment with TL. A recent study indicated that overexpression of c‑Myc in colon cancer cells promotes increased expression of HIF‑1α and VEGF. Therefore, TL may suppress HIF‑1α through a c‑Myc‑dependent mechanism, which is involved in antitumor effects in mouse models of pancreatic cancer.

It is well established that homocysteine (Hcy) is an independent risk factor for atherosclerosis (AS), which is characterized by vascular smooth muscle cell (VSMC) proliferation. However, the molecular mechanism underlying AS in VSMCs is yet to be elucidated. The aim of this study was to investigate the potential involvement of aberrant DNA methylation of the platelet‑derived growth factor (PDGF) gene in Hcy‑mediated VSMC proliferation and its underlying mechanism. Cultured human VSMCs were treated with varying concentrations of Hcy. VSMC proliferation, PDGF mRNA and protein expression and PDGF promoter demethylation showed a dose‑dependent increase with Hcy concentration, suggesting an association among them. Cell cycle analysis revealed a decreased proportion of VSMCs in G0/G1 and an increased proportion in S phase, indicating that VSMC proliferation was increased under Hcy treatment. Furthermore, S‑adenosylhomocysteine (SAH) levels were observed to increase and those of S‑adenosylmethionine (SAM) were observed to decrease. The consequent decrease in the ratio of SAM/SAH may partially explain the hypomethylation of PDGF with Hcy treatment. Folate treatment exhibited an antagonistic effect against Hcy‑induced VSMC proliferation, aberrant PDGF methylation and PDGF expression. These data suggest that Hcy may stimulate VSMC proliferation through the PDGF signaling pathway by affecting the epigenetic regulation of PDGF through the demethylation of its promoter region. These findings may provide novel insight into the molecular association between aberrant PDGF gene demethylation and the proliferation of VSMCs in Hcy‑associated AS.

Von Hippel-Lindau (VHL) disease is an autosomal dominant familial cancer syndrome. VHL is characterized by the development of renal cell carcinoma (RCC), hemangioblastomas of the central nervous system or retina and pheochromocytoma (PCC). RCC and PCC are known to be caused by germline mutations of six and ten genes, respectively. In the present study, 30 individuals from two unrelated pedigrees with type 2A and 2C VHL syndrome were investigated. The patients were clinically examined and treated by radical nephrectomy [or nephron‑sparing surgery (NSS)] and cortical-sparing adrenalectomy (CSA), and all members of the two families underwent genetic screening. Two members from the first family were diagnosed with PCC and RCC, and three individuals from the second family who had only hypertension were diagnosed with PCC. Heterozygous variants of the VHL gene, c.A233G (p.N78S) within exon 1 and c.G482A (p.R161Q) within exon 3, were verified, respectively. Surgery was performed on all the patients, with the exception of an asymptomatic 5-year-old p.N78S male in family 1, in addition to genetic testing and genetic counseling. Further patient follow-up was warranted with regard to blood pressure and health, although normal blood pressure and no local recurrence and distant metastasis of VHL were observed previously. The present study suggests that molecular genetic testing may aid the diagnosis and clinical management of VHL syndrome.

Triggering receptor expressed on myeloid cells-2 (TREM-2) has been shown to attenuate inflammatory responses in various cell lines including bone marrow-derived macrophages, hepatic macrophages, osteoclasts and dendritic cells. However, its effects on alveolar macrophages remain unknown. Lentivirus-mediated RNA interference (RNAi) is a post-transcriptional gene silencing method, which is capable of degrading target genes specifically and efficiently. In this study, we silenced TREM-2 in murine alveolar macrophages by using lentivirus-mediated short hairpin RNA (shRNA) and evaluated the effects of TREM-2 silencing on expression of toll-like receptor-4 (TLR-4), tumor necrosis factor-α (TNF-α) and interleukin-10 (IL-10) in response to lipopolysaccharide (LPS). Alveolar macrophages were transfected with shRNA targeting TREM-2 by use of lentivirus vector, non-sense shRNA as a negative control or empty lentivirus vector as a blank control. Silencing of TREM-2 was assessed by real‑time fluorescence quantitative PCR and flow cytometry. Following LPS stimulation, the levels of TLR-4, TNF-α and IL-10 expressed in alveolar macrophages were measured by real-time PCR, flow cytometry or ELISA. TREM-2 expression on alveolar macrophages was downregulated significantly by lentivirus-mediated shRNA treatment at the transcriptional and translational levels. However, alveolar macrophages that received non-sense shRNA or empty lentivirus vectors showed no effects on TREM-2 expression. Silencing of TREM-2 enhanced expression of TLR-4, as well as TNF-α and IL-10, by alveolar macrophages following LPS stimulation. These results indicate a significant effect of TREM-2 on attenuating the LPS-induced inflammatory response of murine alveolar macrophages, which may be dependent on TLR-4.

The aim of this study was to investigate the effects of emodin, the major component of Rheum palmatum, on lipopolysaccharide (LPS)-induced toll-like receptor 4 (TLR4) expression in cultured mouse tubular epithelial cells (TECs). The TECs were obtained from mice and incubated with LPS and/or indicated concentrations of emodin for 24 h. Cytokeratin, α-SMA and vimentin were detected using immunohistochemistry. The TLR4 protein level was detected by flow cytometry. TNFα and IL-6 protein levels were measured using an enzyme-linked immunosorbent assay (ELISA). mRNA expression of TLR4, TNFα and IL-6 was detected using a reverse-transcription polymerase chain reaction (RT-PCR). Results showed that a concentration of 102 ng/ml LPS significantly upregulated TLR4 mRNA and protein levels. TNFα and IL-6 mRNA and protein levels were also increased. Emodin (at doses of 40, 20 and 10 µM) was able to inhibit LPS-induced TLR4 protein synthesis in cultured TECs. However, TNFα and IL-6 protein expression was decreased in cells treated with emodin at concentrations of 40 and 20 µM. These results demonstrate that an elevated expression of inflammatory cytokines and TLR4 in cells stimulated with LPS, were simultaneously inhibited by emodin. Emodin is therefore able to inhibit the LPS-induced expression of TLR4 in order to downregulate TNFα and IL-6 synthesis in TECs, which may contribute to the protective effects of emodin in renal disease.

Emodin has been demonstrated to inhibit the fibrotic process in chronic renal disease, but its mechanisms have yet to be fully elucidated. This study was carried out to investigate the effects of emodin on extracellular matrix (ECM) synthesis in TGF-β1-stimulated NRK-49F cells. NRK-49F cells stimulated with TGF-β1 were incubated with various concentrations of emodin. ECM proteins, including collagen type III and fibronectin, were detected using ELISA. ERK1/2, p38 and JNK phosphorylation were measured by Western blotting. p38, ERK1/2 and JNK were respectively inhibited with the specific inhibitors SB203580, PD98059 and SP600125. Emodin slightly inhibited the expression of fibronectin and collagen type III in NRK-49F cells without TGF-β1 treatment, and significantly suppressed fibronectin and collagen type III secretion in TGF-β1-stimulated NRK-49F cells. ERK1/2 and p38 specific inhibitors, but not JNK inhibitor, suppressed the TGF-β1-induced expression of fibronectin and collagen type III. Our previous study demonstrated that there was no crosstalk between ERK1/2, p38 and JNK signals in TGF-β1-stimulated NRK-49F cells. Here, we found that emodin inhibited the phosphorylation of ERK1/2 and p38 significantly, but did not suppress the phosphorylation of JNK. In summary, emodin suppresses fibronectin and collagen type III expression via the inhibition of ERK1/2 and p38 phosphorylation in TGF-β1-stimulated NRK-49F cells.