Rab5a, a key member of the Rab family of GTPases, was determined to be a regulator of vascular smooth muscle cell (VSMC) proliferation and migration. However, the exact regulatory mechanism remains unclear. As Rab5a has been shown to be associated with autophagy, which is essential for the conversion of VSMCs from a contractile to a synthetic phenotype in order to prevent cell death due to oxidative stress. The present study hypothesized that autophagy may be responsible for the proliferation and migration of VSMCs via the Rab5a protein. The aim of the present study was to evaluate the effect of Rab5a on autophagy in VSMCs. The human aorta vascular smooth muscle cell line, T/G HA‑VSMCs, was treated with small interfering (si)RNA against Rab5a and/or platelet‑derived growth factor (PDGF). Following treatment, the phenotype transition of the VSMCs was evaluated by detecting the mRNA and protien expression levels of VSMC molecular markers using reverse transcription‑quantitative polymerase chain reaction and western blotting, respectively. In addition, autophagy in VSMCs was evaluated by western blotting for autophagy‑associated proteins, flow cytometry of acidic vesicular organelles, punctate fluorescence of microtubule associated protein light chain 3 and transmission electron microscopy of typical scattered double‑membrane vacuolar structures. Additionally, the proliferation, migration, cell cycle and apoptotic response of VSMCs were detected by sulforhodamine B assay, transwell assay and flow cytometry, respectively. The results revealed that transfection with siRNA against Rab5a led to a significant decrease in Rab5a protein expression, while the reduced expression trend of Rab5a was rescued by intervention with PDGF. Furthermore, cells transfected with siRNA against Rab5a inhibited the autophagy of VSMCs. Downregulated Rab5a inhibited the phenotype transition of VSMCs. Additionally, downregulated Rab5a led to slowed cell growth, decreased numbers of migrated cells, decreased numbers of cells at the G0‑G1 phase and a higher apoptosis rate. However, PDGF significantly rescued these phenomena caused by siRNA against Rab5a. These results indicated that Rab5a‑mediated autophagy may regulate the phenotype transition and cell behavior of VSMCs through the activation of the extracellular‑regulated kinase 1/2 signaling pathway.

The present study aimed to identify the genes and underlying mechanisms critical to the pathology of spinal cord injury (SCI). Gene expression profiles of spinal cord tissues of trkB.T1 knockout (KO) mice following SCI were accessible from the Gene Expression Omnibus database. Compared with trkB.T1 wild type (WT) mice, the differentially expressed genes (DEGs) in trkB.T1 KO mice following injury at different time points were screened out. The significant DEGs were subjected to function, co‑expression and protein‑protein interaction (PPI) network analyses. A total of 664 DEGs in the sham group and SCI groups at days 1, 3, and 7 following injury were identified. Construction of a Venn diagram revealed the overlap of several DEGs in trkB.T1 KO mice under different conditions. In total, four modules (Magenta, Purple, Brown and Blue) in a co‑expression network were found to be significant. Protein tyrosine phosphatase, receptor type C (PTPRC), coagulation factor II, thrombin (F2), and plasminogen (PLG) were the most significant nodes in the PPI network. 'Fc γ R‑mediated phagocytosis' and 'complement and coagulation cascades' were the significant pathways enriched by genes in the PPI and co‑expression networks. The results of the present study identified PTPRC, F2 and PLG as potential targets for SCI treatment, which may further improve the general understanding of SCI pathology.

The effector function of natural killer, lymphokine--activated killer cells and T lymphocytes is commonly evaluated by radioactive chromium‑release cytotoxicity assays. In addition to this indirect method, fluorescence assays have been described for the assessment of in vitro cell‑mediated cytotoxicity. In the present study, target cells were stained with 5‑(and‑6)‑carboxyfluorescein diacetate succinimidyl ester (CFSE), which is a stable integrated fluorescent probe that allows target and effector cells to be distinguished from one another. Staining of target THP‑1 cells with 8 µM CFSE revealed high and stable loading of fluorescence and no effect of the viability of cells. After 4 h of in vitro co‑culture between γδ T cells and CFSE‑labeled infected or uninfected THP‑1 cells, staining with propidium iodide (PI) was performed to distinguish between vital and dead cells. During sample acquisition, target cells were gated on the CFSE positivity and examined for cell death based on the uptake of PI. CFSE and PI double positive cells were recognized as the dead target cells. The percentage of cytotoxicity in the CFSE‑gated cell population was calculated by subtracting the value obtained for non‑specific PI‑positive target cells, which was measured in a control group that did not contain effector cells. The present study describes a simple and convenient assay that is based on the direct quantitative and qualitative analysis of cell damage at a single cell level utilizing a two‑color flow cytometric assay. In conclusion, the flow cytometric‑based assay described in the current study is a simple, sensitive and reliable tool to determine the cytolytic activity of γδ T lymphocytes against mycobacteria. Therefore, the present study may provide valuable information concerning the methods employed to investigate the function of γδ T cells and potentially other lymphocyte subsets.

Ethanol has a toxic effect on the heart, resulting in cardiomyocyte damage. Long‑term high intake of ethanol leads to a non‑ischemic dilated cardiomyopathy termed alcoholic cardiomyopathy (ACM). However, the pathogenesis of alcoholic cardiomyopathy remains unclear. The apoptosis of cardiomyocytes serves an important role in the pathogenesis of ACM. X‑linked inhibitor of apoptosis protein (XIAP) is an important anti‑apoptotic protein in human tissue cells. To the best of our knowledge, no studies have reported on its function in ethanol‑induced cardiomyopathy. Previous works have screened the ACM‑associated differentially expressed microRNAs (miRs), including miR‑186‑5p and miR‑488‑3p. TargetScan bioinformatics software was used to predict 949 target genes associated with miR‑186‑5p, and XIAP was demonstrated to be a target of miR‑186‑5p. The present study firstly analyzed the levels of apoptosis in ethanol‑treated cardiomyocytes using flow cytometry. Alterations in the expression levels of miR‑186‑5p and XIAP were subsequently evaluated in ethanol‑treated AC16 cardiomyocytes to assess the specific molecular mechanisms of ethanol‑induced cardiomyocyte apoptosis. The levels of apoptosis in AC16 cardiomyocytes increased following ethanol treatment, and further increased with the rise in concentration and action time of ethanol. The expression levels of miR‑186‑5p were upregulated, and the expression levels of XIAP were downregulated in ethanol‑treated cardiomyocytes. miR‑186‑5p may regulate ethanol‑induced apoptosis in cardiomyocytes using XIAP as the direct target gene. This study provides a novel therapeutic target for the prevention and treatment of ACM.

The present study aimed to investigate protein expression levels of intra‑ and extracranial atherosclerosis in rabbits following administration of a high‑fat diet. Rabbits were randomly divided into control (group A; n=9) and high‑fat diet (group B; n=9) groups. At week 12, tissues were sectioned from the common carotid artery (CCA) and middle cerebral artery (MCA). Pathological analysis was performed. Differential protein expression levels were examined by 2‑D gel electrophoresis (2‑DE) and mass spectrometry (MS) analysis and validated by western blotting. Serum lipid levels, the intima‑media thickness (IMT) and degree of atherosclerosis of the CCA and MCA were increased at week 12 in the high‑fat diet group compared with rabbits that received a normal diet. 2‑DE and MS analysis of the protein extracted from CCA and MCA detected >439 different proteins; the expression of 25 proteins was altered, and 8 proteins [albumin A chain, tropomyosin α‑1 chain (TPM1), heat shock protein 70 (HSP70), α‑smooth muscle actin, β‑galactose binding agglutinin, TPM4 isoform 2, cell keratin 9, single octylic acid glyceride β‑2) demonstrated significant alterations in expression levels. Due to limited antibody sources, only three differentially expressed proteins (TPM1, HSP70 and α‑smooth muscle actin) were examined by western blotting. The results of our previous study demonstrated that hyperlipidemia affected the IMT of intracranial and extracranial cerebral arteries. In the present study, protein expression levels of TPM1 and α‑smooth muscle actin from extracranial cerebral arteries were significantly increased compared with intracranial cerebral arteries; however, protein expression levels of HSP70 from intracranial cerebral arteries was increased compared with extracranial cerebral arteries. The differences may be closely associated with cell proliferation and metastasis, and oxidoreduction, in intra‑ and extracranial cerebral atherosclerosis. HSP70 may have protective properties against atherosclerosis via underlying anti‑inflammatory mechanisms, furthermore, differential protein expression levels (TPM1, HSP70 and α‑smooth muscle actin) between intra‑ and extracranial cerebral arteries may facilitate the identification of novel biological markers for the diagnosis and treatment of cerebral arteriosclerosis.

Bone marrow‑derived mesenchymal stem cells (BM‑MSCs) have been used in experimental research and clinical trials for heart function restoration and cardiomyocyte regeneration. However, due to a hostile microenvironment created by ischemia, hypoxia and pro‑inflammatory factors, the survival rate of implanted BM‑MSCs remains low. Therefore, strategies that can promote BM‑MSC survival and prevent apoptosis are required. Previous studies have reported that microRNA‑16 (miR‑16) can inhibit cell proliferation by targeting several proteins and signal pathway, not only by inducing apoptosis. In the present study, it was investigated whether inhibition of miR‑16 reduced BM‑MSC apoptosis in a model of ischemia. Flow cytometry analysis revealed that BM‑MSCs underwent apoptosis in response to hypoxia/serum deprivation (SD). Additionally, in hypoxic/SD conditions, miR‑16 expression increased and B‑cell lymphoma (Bcl)‑2 protein expression decreased in BM‑MSCs. miR‑16 did not affect Bcl‑2 mRNA expression but downregulated Bcl‑2 protein expression. miR‑16 inhibitor transfection significantly increased Bcl‑2 protein expression and the percentage of apoptotic BM‑MSCs was reduced. The pro‑apoptotic effects of miR‑16 were partially elevated by knocking down of Bcl‑2. Furthermore, it was demonstrated that miR‑16 exerted its pro‑apoptotic effects by activating the mitochondrial pathway of apoptosis via apoptotic protease activating factor‑1/caspase‑9/poly (ADP ribose) polymerase. Taken together, the results indicated that miR‑16 downregulated Bcl‑2 expression and promoted BM‑MSC apoptosis, indicating that therapies targeting miR‑16 may improve the effectiveness of BM‑MSC transplantation therapy.

Dendritic cells (DCs) serve crucial roles in the activation of the immune response, and imbalance in the activation or inhibition of DCs has been associated with an increased susceptibility to develop immune‑induced diseases. However, the molecular mechanisms of regulating immune‑induced diseases of DCs are not well understood. The aim of the present study was to identify the gene signatures and uncover the potential regulatory mechanisms in DCs. A total of 4 gene expression profiles (GSE52894, GSE72893, GSE75938 and GSE77969) were integrated and analyzed in depth. In total, 241 upregulated genes and 365 downregulated genes were detected. Gene ontology and pathway enrichment analysis showed that the differentially expressed genes (DEGs) were significantly enriched in the inflammatory response, the tumor necrosis factor (TNF) signaling pathway, the nuclear factor (NF)‑κB signaling pathway and antigen processing. The top 10 hub genes were identified from the protein‑protein analysis. The most significant 2 modules were filtered from the protein‑protein network. The genes in 2 modules were involved in type I interferon signaling, the NF‑κB signaling pathway and the TNF signaling pathway. Furthermore, the microRNA‑mRNA network analysis was performed. The results of the present study revealed that the identified DEGs and pathways may improve our understanding of the mechanisms of the maturation of DCs, and the candidate hub genes that may be therapeutic targets for immune‑induced diseases.

MicroRNAs (miRNAs) are non‑coding RNAs of ~22 nucleotides in length, which serve an important role in numerous diseases. Asthma is a chronic airway inflammatory disease, which is the most common chronic disease among children. The role of miRNA (miR)‑16 in asthma is unclear. The objective of the present study was to examine the underlying molecular mechanism of the involvement of miR‑16 in asthma. A total of 72 volunteers diagnosed with asthma consented to participate in the study, of whom 52 participants were identified to be sensitive to salmeterol and 20 participants were identified to be resistant to salmeterol. Receiver operating characteristic (ROC) curve analysis was performed to compare the expression levels of serum miR‑16 between the sensitive and resistant groups, and to confirm the association between the expression level of serum miR‑16 and forced expiratory volume in 1 sec (FEV1). In silico analysis, a luciferase assay, reverse transcription‑quantitative polymerase chain reaction analysis and western blotting were performed to elucidate the molecular mechanism underlying the role of miR‑16 in asthma. ROC results demonstrated that the serum miR‑16 level may function as a biomarker to predict the response to salmeterol therapy, and the miR‑16 expression level displayed a significant negative correlation with FEV1. According to the in silico analysis, adrenoreceptor β‑2 (ADRB2) was a direct target of miR‑16, and it was further confirmed by luciferase assay that 25 nM miR‑16 mimic had an inhibitory effect on the luciferase activity of the wild‑type ADRB2 3' untranslated region (UTR); the inhibitory effect on the luciferase activity of the wild‑type ADRB2 3'UTR was stronger with 50 nM miR‑16 mimic, and strongest with 75 nM miR‑16 mimic, whereas the luciferase activity of the mutant ADRB2 3'UTR in cells was similar following treatment with 0, 25, 50 or 75 nM miR‑16 mimic. miR‑16 reduced the mRNA and protein expression levels of ADRB2 in a dose‑dependent manner. These results identified that miR‑16 may be used as a predictive biomarker of therapeutic response in asthma.

Long non‑coding RNA small nucleolar RNA host gene 12 (SNHG12) has been demonstrated to be oncogenic. The aim of the present study was to examine the effects of SNHG12 on the progression of endometrial cancer (EC). The expression levels of SNHG12 and microRNA (miR)‑4429 were assessed in EC cell lines by reverse transcription‑quantitative PCR. Plasmids, including SNHG12 short hairpin RNAs (shRNAs), shRNA negative control (NC), SNHG12 overexpression (OV), OV‑NC, miR‑4429 mimic and mimic‑NC, were transfected into RL95‑2 cells. Post‑transfection, Cell Counting Kit‑8, Transwell Matrigel and wound‑healing assays were performed to assess cell proliferation, invasion and migration, respectively. Cell cycle phase distribution was assessed by flow cytometry. The protein expression levels of matrix metalloproteinase (MMP)2 and MMP9 were detected by western blotting. miR‑4429 target genes were predicted by bioinformatics analysis using target prediction online tools; the findings of this analysis were verified using a dual‑luciferase reporter system. Identified as a target of miR‑4429, SNHG12 was overexpressed in EC cell lines with decreased expression of miR‑4429. Further experiments demonstrated that SNHG12 silencing and overexpression of miR‑4429 markedly suppressed proliferation, migration and invasion of RL95‑2 cells, arrested cells in the G1 phase, and markedly downregulated the expression of MMP2 and MMP9. The opposite effects were observed in miR‑4429 mimic‑transfected RL95‑2 cells after SNHG12 was overexpressed. The findings of the present study established the role of SNHG12 and miR‑4429 in EC. Therefore, targeting the SNHG12/miR‑4429 axis could serve as a potential future therapeutic target for treatment of EC.

The modification of Wnt and Notch signaling pathways by hypoxia, and its association with osteoblast proliferation and apoptosis remain to be fully elucidated. To investigate Wnt-Notch crosstalk, and its role in hypoxia-induced osteoblast proliferation and apoptosis regulation, the present study investigated the effects of cobalt‑mimicked hypoxia on the mouse pre-osteoblast-like cell line, MC3T3‑E1, when the Notch signals were repressed using a γ‑secretase inhibitor DAPT. The data showed that the cobalt‑mimicked hypoxia suppressed cell proliferation under normal conditions, but increased cell proliferation under conditions of Notch repression, in a concentration‑dependent manner. The results of western blot and reverse transcription‑quantitative polymerase chain reaction analyses showed that the cobalt treatment increased the levels of activated β‑catenin protein and the expression levels of the target genes, axis inhibition protein 2 and myelocytomatosis oncogene, under DAPT‑induced Notch repression. However, no significant changes were found in the expression levels of the Notch intracellular domain protein or the Notch target gene, hes1. In a β‑catenin gene‑knockdown experiment, the proliferation of the MC3T3‑E1 cells under hypoxia were decreased by DAPT treatment, and knockdown of the expression of hypoxia‑inducible factor‑1α (HIF‑1α) suppressed the cobalt‑induced increase in Wnt target gene levels. No significant difference in cell proliferation rate was found following DAPT treatment when the expression of HIF‑1α was knocked down. The results of the present study showed the opposing effects of Wnt and Notch signaling under cobalt‑mimicked hypoxia, which were partially regulated by HIF‑1α, The results also showed that osteoblast proliferation was dependent on Wnt-Notch signal crosstalk.

Hypermethylation of transcription factor activating enhancer‑binding protein 2e (TFAP2E) has been reported to be associated with chemoresistance to 5‑fluorouracil (5‑FU) in gastric cancer (GC). In the present study, the molecular mechanism governing this chemoresistance was investigated. Drug‑resistant human GC MGC‑803/5‑FU cells were established and TFAP2E expression and methylation levels were assessed. Autocrine exosomes from GC culture medium were isolated and characterized. MicroRNA (miRNA) microarray analysis was used to determine the miRNA expression profile of GC cell‑derived exosomes. Exosomes collected from MGC‑803/5‑FU cells were co‑cultured with control cells, and 5‑Aza‑2'‑deoxycytidine (5Aza) was added into MGC‑803/5‑FU cells to investigate the relationship between TFAP2E, exosomes and chemosensitivity. In the present study, it was demonstrated that hypermethylation of TFAP2E resulted in its reduced expression and 5‑FU chemoresistance in GC cells. miRNAs miR‑106a‑5p and miR‑421 were highly expressed and regulated the chemoresistance induced by TFAP2E methylation. Target gene prediction using miRBase, TargetScan and PicTar revealed that E2F1, MTOR and STAT3 may be TFAP2E target genes in GC. Collectively, our data support an important role of exosomes and exosomal miRNAs in TFAP2E methylation‑induced chemoresistance to 5‑FU in GC. These results highlight their potential for miRNA‑based therapeutics.