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Gingival squamous cell carcinoma (GSCC) is responsible fora large proportion of oral cavity malignancies. GSCC is characterized by rapid cell growth, and progressive invasion and migration. P21 is a widely recognized tumor suppressor, which is induced by P53 activation; however, drugs that aim to promote P21-mediated tumor suppression remain to be identified. A natural compound library was used to perform broad-spectrum screening of drugs that could promote P21 expression. Subsequently, the effects of the screened drug on GSCC cell proliferation and apoptosis were evaluated. The results of the present study suggested that lapiferin was the most effective natural compound that promoted the expression of P21 at both mRNA and protein levels. Lapiferin inhibited proliferation and enhanced apoptosis of YD-38 GSCC cells in a dose-dependent manner. Furthermore, following treatment with lapiferin, the critical cell cycle regulators cell division cycle 25C and cyclin B1 and tumor cell proliferation markers proliferating cell nuclear antigen and Ki67 were markedly decreased. In addition, pro-apoptotic proteins were promoted following treatment of YD-38 cells with lapiferin. Following the depletion of P21 expression, lapiferin-mediated inhibition of cell proliferation and enhancement of cell apoptosis were significantly reduced. These results indicated that lapiferin may exert potent antitumor effects on GSCC via regulation of P21; therefore, lapiferin may be considered a potential, natural therapeutic agent for the treatment of GSCC.

Our previous study reported that fully reduced high mobility group box 1 (fr‑HMGB1) and disulfide HMGB1 (ds‑HMGB1) induce depressive‑like behavior; however, the underlying mechanisms remain unclear. In the present study, the induction of depression via the kynurenine pathway by different redox states of HMGB1 was investigated in vivo and in vitro. To evaluate the expression of enzymes of the kynurenine pathway, reverse transcription‑quantitative PCR and western blot analyses were conducted. Additionally, cytokine levels were measured by ELISAs. Following intracerebroventricular injection of ds‑ and fr‑HMGB1, behavioral tests were performed, revealing the presentation of depressive‑like behavior, and essential proteins in the kynurenine pathway were demonstrated to be upregulated at the mRNA level, suggesting that ds‑ and fr‑HMGB1 contributed to the development of this behavior via the kynurenine pathway. ds‑HMGB1 directly activated the kynurenine pathway and cytokines such as tumor necrosis factor‑α (TNF‑α) and interleukin‑1β (IL‑1β) in the hippocampal tissue. Conversely, fr‑HMGB1 upregulated the aforementioned factors only following treatment with H2O2. These findings indicated that ds‑HMGB1 induced depression in a manner associated with the kynurenine pathway, whereas oxidation of fr‑HMGB1 evoked activation of the kynurenine pathway, resulting in depressive behavior.

The clinical symptoms of chronic hepatitis B virus (HBV) infection include severe liver damage, which is associated with the elimination of the HBV‑infected cells by the immune system. It has been suggested that suppression of HBV replication is not sufficient for patients with hepatitis B and the damaged liver function requires restoration. In the present study, mesenchymal stem cells (MSCs) were combined with short hairpin (sh)RNA to treat liver injury and suppress HBV replication in a mouse model. Lx‑shRNA157‑1694 (an shRNA expression plasmid containing two shRNA expression cassettes) and mouse immortal (mi)MSCs stably expressing shRNA (miMSC‑shRNA) were constructed and their suppressive effects on HBV expression were investigated using reverse transcription-polymerase chain reaction (RT‑PCR), ELISA and immunofluorescence. Hepatogenic differentiation of miMSC‑shRNA was induced in vitro and confirmed by morphology, reverse transcription‑semi‑quantitative and ‑quantitative PCR, urea production and Periodic acid‑Schiff staining analyses. miMSCs and the shRNA expression plasmid alone or combined with miMSCs stably expressing shRNA were injected into mice. The former therapeutic regimen successfully suppressed HBV expression in sera and liver tissue, whereas the latter only suppressed HBV expression in liver tissue. Analyses of serum alanine aminotransferase levels, aspartate aminotransferase levels, liver weight/body weight ratio percentage and sirius red staining demonstrated marked amelioration of liver injury in mice treated with both therapeutic regimens. The results of the present study suggest that miMSCs combined with shRNA treatment may alleviate liver injury and suppress HBV expression, thus providing a novel potential therapeutic strategy for the treatment of liver injury induced by HBV infection.

The discovery of cell-free DNA fetal (cff DNA) in maternal plasma during pregnancy provides a novel perspective for the development of non-invasive prenatal diagnosis (NIPD). Against the background of maternal DNA, the use of the relatively low concentration of cff DNA is limited in NIPD. Therefore, in order to overcome the complication of the background of maternal DNA and expand the scope of cff DNA application in clinical practice, it is necessary to identify novel universal fetal-specific DNA markers. The GeneChip Human Promoter 1.0R Array set was used in the present study to analyze the methylation status of 12 placental tissue and maternal peripheral blood whole-genome DNA samples. In total, 5 fetus differential hypermethylation regions and 6 fetus differential hypomethylation regions were identified. In order to verify the 11 selected methylation regions and detect the differential CpG sites in these regions, a bisulfate direct sequencing strategy was used. In total, 87 fetal differential methylation CpG sites were identified from 123 CpG sites. The detection of fetal differential methylation DNA regions and CpG sites may be instrumental in the development of efficient NIPD and in the expansion of its application in other disorders.

Retinitis pigmentosa (RP) is a common form of inherited retinal degeneration that causes progressive loss of vision or adult blindness, characterized by the impairment of rod and cone photoreceptors. At present, mutations in >60 pathogenic genes have been confirmed to cause RP. The predominant modes of inheritance are autosomal dominant, autosomal recessive and X-linked. In addition, other modes of inheritance, including digenic or mitochondrial inheritance, have been reported. In previous decades, with the development of sequencing techniques, significant advances in identifying novel RP pathogenic genes and screening mutations have been made. In the present study, whole-exome sequencing was performed on samples from two Chinese pedigrees diagnosed with RP. A compound heterozygous mutation in the gene usherin 2A (USH2A; c.6,485+5G>A/c.11,156G>A) and a heterozygous X-linked mutation in the gene retinitis pigmentosa 2 (RP2) ARL3 GTPase-activating protein (RP2; c.358C>T) were identified by Sanger sequencing and co-segregation analysis, of which the pathogenic mutation (c.6,485+5G>A) in USH2A has not been previously reported among Chinese patients. The findings of the present study may expand on current knowledge of RP among the Chinese population, providing essential assistance in the molecular diagnosis and screening of RP, and promoting further investigation of the pathogenesis of RP.

Congenital heart disease (CHD), and cleft lip and palate (CLP) are currently the most common types of structural malformation in infants. Various methods have been used to identify the disease‑associated genes. However, targeted next‑generation sequencing (NGS) is not yet considered an option for routine use. Thus, the present study aimed to assess the safety and feasibility of using targeted NGS in patients with CHD concomitant with CLP. Between November 2015 and May 2017, a total of 17 patients with CHD concomitant with CLP, who were excluded from a diagnosis of trisomy syndrome, were selected at The Second Xiangya Hospital of Central South University (Changsha, China). Genomic DNA was extracted from peripheral blood samples of the patients. The copy number variants (CNVs) were determined by conducting a single nucleotide polymorphism (SNP) array with Illumina HumanOmni1‑Quad Beadchip, while information on other gene mutations was obtained from targeted sequencing. The functions of gene mutations were then predicted using the PolyPhen‑2, SIFT and Mutation Taster tools. Finally, Sanger sequencing was used to verify the mutations. The results identified no pathogenic mutations in CNVs analyzed by high‑throughput SNP sequencing. Targeted NGS results demonstrated that 10 patients (58.8%) carried gene mutations, including 4 (23.5%) genetically diagnosed cases and 6 (35.3%) cases with unknown etiology. The 4 known diseases were Opitz G/BBB syndrome caused by MID1 gene mutation, Loeys‑Dietz syndrome caused by TGFBR1 gene mutation, Ritscher‑Schinzel/3C syndrome caused by KIAA0196 gene mutation and CHARGE syndrome caused by CHD7 gene mutation. The remaining 6 cases were not genetically diagnosed, although they carried candidate genes. In conclusion, the present study demonstrated that targeted NGS was an effective and accurate candidate gene detection method in patients with CHD concomitant with CLP.

Mesenchymal stem cells (MSCs) with the ability to differentiate into insulin‑producing cells (IPCs) have become the most promising means of therapy for diabetes mellitus. Adipose‑derived stromal cells (AdSCs), having similar characteristics to those of derived MSCs, are known to exhibit extensive proliferation potential and are able to undergo multi‑lineage differentiation. Whether AdSCs can differentiate into insulin‑producing cells (IPCs), however, has not been sufficiently elucidated. Therefore, the present study sought to investigate the in vitro differentiation of rabbit (r)AdSCs into IPCs, which may provide an abundant source of cells to treat diabetes. rADSCs were obtained from liposuction aspirates and then induced with glucagon‑like peptide‑1 and nicotinamide to differentiate into insulin‑secreting cells. Differentiation was evaluated by the analysis of morphology, dithizone (DTZ) staining, reverse transcription polymerase chain reaction (RT‑PCR), western blot analysis and a glucose challenge assay with detection of insulin secretion by ELISA. Morphological phase‑contrast microscopic observation revealed typical islet‑like cell clusters following 21 days of differentiation. DTZ staining also showed that differentiated cells were positive and undifferentiated cells were negative for insulin production. Furthermore, RT‑PCR analysis confirmed the mRNA expression of insulin, PDX1 and GLUT2 in differentiated cells. Western blot analysis showed that insulin was expressed by the differentiated cells. The glucose challenge assay showed that insulin secretion of the IPCs was in a glucose dependent manner. These findings implied that AdSCs are able to differentiate into IPC in vitro, and are therefore promising candidates for the treatment of diabetes.

DL0410, a dual-action cholinesterase inhibitor and histamine-3 receptor antagonist with a novel structural scaffold, may be a potential candidate for the treatment of Alzheimer's disease (AD). To the best of the authors' knowledge, this is the first study to demonstrate a reliable method for the measurement of DL0410 in rat plasma, brain, bile, urine and feces samples, and identification of its primary metabolites. The pharmacokinetic properties of DL0410 were analyzed by liquid chromatography-mass spectrometry at oral doses of 25, 50 and 100 mg/kg and intravenous dose of 5 mg/kg. The investigation of the excretion and metabolism of DL0410 was determined following liquid-liquid extraction for biliary, urinary and fecal samples. Finally, the cytochrome (CY)P450 isoforms involved in the production of DL0410 metabolites with recombinant human cytochrome P450 enzymes were characterized. The results suggested that DL0410 was not well absorbed; however, was distributed to the entorhinal cortex and hippocampus of the brain. A total of two common metabolites of the reduction of DL0140 in the bile, urine and feces were identified and CYP2D6 was involved in this reaction. The pharmacokinetic results of DL0410 provided information for the illustration of its pharmacodynamic properties, mechanism of action and promoted its continued evaluation as a therapeutic agent for AD treatment.

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.

Chronic administration of D‑galactose (D‑gal) is a useful method for establishing a model of natural aging in the auditory system. Previous studies have demonstrated that NADPH oxidases (NOXs) may be an important source of reactive oxygen species (ROS) in the peripheral auditory system (PAS) and cause an increase in mitochondrial DNA (mtDNA) common deletion (CD) levels in the PAS and central auditory system (CAS) of rats with D‑gal‑induced aging. However, the source of the ROS in the CAS and the mechanisms of age‑related hearing loss (ARHL) have yet to be elucidated. In the present study, male Sprague Dawley rats were administered a daily injection of D‑gal (150, 300 and 500 mg/kg, respectively) for eight weeks. All three doses of D‑gal caused a significant increase in the expression of NOX2, 8‑hydroxy‑2‑deoxyguanosine, a biomarker of DNA oxidative damage, and uncoupling protein 2, together with a decrease in the mitochondrial total antioxidant capabilities in the auditory cortex, as compared with the control rats (injected daily with the same volume of 0.9% saline for eight weeks). The levels of the mtDNA CD were also increased in the auditory cortex of the D‑gal‑induced aging rats. These findings suggest that both NOX‑ and mitochondria‑associated ROS generation may contribute to mtDNA oxidative damage in the auditory cortex of the CAS of D‑gal‑induced aging rats. This study may provide novel insight into the development of ARHL.

Optic neurodegeneration, in addition to central nervous trauma, initiates impairments to neurons resulting in retinal ganglion cell (RGC) damage. Carbon monoxide (CO) has been observed to elicit neuroprotection in various experimental models. The present study investigated the potential retinal neuroprotection of preconditioning with CO inhalation in a rat model of optic nerve crush (ONC). Adult male Sprague‑Dawley rats were preconditioned with inhaled CO (250 ppm) or air for 1 h prior to ONC. Animals were euthanized at 1 or 2 weeks following surgery. RGC densities were quantified by hematoxylin and eosin (HE) staining and FluoroGold labeling. Visual function was measured via flash visual evoked potentials (FVEP). Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, and caspase‑9 and caspase‑3 activity in the retinas, were assessed at 2 weeks post‑ONC. The RGC density of CO + crush rats was significantly increased compared with that of the corresponding crush‑only rats at 2 weeks (survival rate, 66.2 vs. 48.2% as demonstrated by HE staining, P0.01; and 67.6 vs. 37.6% as demonstrated by FluoroGold labeling, P0.05). FVEP measures indicated a significantly better‑preserved latency and amplitude of the P1 wave in the CO + crush rats compared with the crush‑only rats. The TUNEL assays demonstrated fewer apoptotic cells in the CO + crush group compared with the crush‑only group, accompanied by the suppression of caspase‑9 and caspase‑3 activity. The results of the present study suggested that inhaled CO preconditioning may be neuroprotective against ONC insult via inhibition of neuronal apoptosis.

The present study aimed to determine the mechanism by which low‑intensity intermittent negative pressure affects the differentiation and proliferation of human mesenchymal stem cells (MSCs). Alkaline phosphatase (ALP) activity, type I collagen and vascular endothelial growth factor (VEGF) were detected to analyze differentiation. MTT and flow cytometry were employed to measure proliferation and apoptosis. Western blot analysis was used to examine endoplasmic reticulum (ER) stress‑associated factors. This study was divided into two groups, including a normal group (without any treatment) and vacuum group (treated with a vacuum). There was a significant decrease in the proliferation of cells in the vacuum group. The number of cells in S phase was reduced significantly, while the rate of apoptosis and the activity of ALP were markedly increased under vacuum conditions. Expression of collagen type I and VEGF was significantly increased, and the ratio of osteoprotegrin to osteoprotegrin ligand was decreased significantly in the vacuum group. ER stress‑associated proteins, p‑PRKR‑like ER kinase, inositol‑requiring enzyme 1 and cleaved activating transcription factor 6, as well as the downstream factors, were activated when treated with negative pressure. In conclusion, treatment with low‑intensity and intermittent negative pressure may inhibit the proliferation of MSCs and trigger ER stress‑associated cellular apoptosis, further enhancing osteogenesis activity and inducing differentiation to osteoblasts.

The present study aimed to identify the mutation causing an atypical syndrome. High-resolution single nucleotide polymorphism (SNP) arrays are considered to be a major detection method for submicroscopic chromosomal rearrangements smaller than 5 Mb in size. Genomic DNA samples of the patient and his parents were converted to a final concentration of 50 ng/ml. The Illumina BeadScan genotyping system and the HumanOmni1‑Quad Chip were employed to obtain the signal intensities of SNP probes. The patient presented with congenital heart disease, autism, mental retardation, growth retardation, hypercalcemia, nephroliths and cleft palate. The karyotypes of the patient and his parents were normal. The present study employed high‑resolution SNP arrays to analyze the whole genome for copy number variations (CNVs). A total of 309 CNVs were discovered. A de novo 1.5 Mb gain of chromosome 7q11.23 (Chr7: 72,357,322‑73,856,472) was identified following exclusion of CNVs presented in the Database of Genomic Variants. In conclusion, to the best of our knowledge, the current study describes the first case of a patient presenting with Williams‑Beuren syndrome alongside supravalvular aortic stenosis, autism and cleft palate, and identifies an atypical deletion at 7q11.23.

Dehydroepiandrosterone (DHEA) is widely used as a nutritional supplement and exhibits putative anti-aging properties. However, the molecular basis of the actions of DHEA, particularly on the biological characteristics of target cells, remain unclear. The aim of the current study was to investigate the effects of DHEA on cell viability, cell proliferation, cell cycle and mitochondrial function in primary rat Leydig cells. Adult Leydig cells were purified by Percoll gradient centrifugation, and cell proliferation was detected using a Click-iT® EdU Assay kit and cell cycle assessment performed using flow cytometry. Mitochondrial membrane potential was detected using JC-1 staining assay. The results of the current study demonstrate that DHEA decreased cell proliferation in a dose-dependent manner, whereas it improved cell viability in a time-dependent and dose-dependent manner. Flow cytometry analysis demonstrated that DHEA treatment increased the S phase cell population and decreased the G2/M cell population. Cyclin A and CDK2 mRNA levels were decreased in primary rat Leydig cells following DHEA treatment. DHEA treatment decreased the transmembrane electrical gradient in primary Leydig cells, whereas treatment significantly increased succinate dehydrogenase activity. These results indicated that DHEA inhibits primary rat Leydig cell proliferation by decreasing cyclin mRNA level, whereas it improves cells viability by modulating the permeability of the mitochondrial membrane and succinate dehydrogenase activity. These findings may demonstrate an important molecular mechanism by which DHEA activity is mediated.

Phorbol myristate acetate (PMA), as a potent tumor promoter, may induce microglial senescence. The present study investigated the effect of PMA infection on microglial senescence. From 58 male Sprague‑Dawley rats, 10 were randomly selected and divided into a PMA injection group, containing five rats (0.5 µg/µl PMA) and a control group, containing five rats (commensurable 0.9% saline). Immunofluorescent staining of Iba‑1 and enzyme‑linked immunosorbent assay analyses of the expression levels of tumor necrosis factor (TNF)‑α and interleukin (IL)‑1 β were performed in these two groups. The remaining 48 rats were randomly divided into the following three groups, each containing 16 rats: Repeated injection control group (commensurable normal saline, once a week for 4 weeks), single PMA injection group (0.5 µg/µl PMA, once in the first week) and repeated injection PMA group (0.5 µg/µl PMA, once a week for 4 weeks). The expression levels of p21, detected using double immunofluorescence staining with Iba‑1, and β‑galactosidase, via double immunohistochemical staining of Iba‑1, were examined in these three groups. The results indicated that a single injection of PMA did not change the microglial morphology and had no significant effects on the expression levels of TNF‑α and IL‑1β, compared with the control group (P>0.05). Following four repeated injections of PMA, the microglia in the substantia nigra presented with features of senescence, characterized by increased expression levels of β-galactosidase (P

The aim of the current study was to investigate disease-associated genes and related molecular mechanisms of osteoarthritis (OA) and rheumatoid arthritis (RA). Using GSE7669 datasets downloaded from Gene Expression Omnibus databases, the differentially expressed genes (DEGs) between RA and OA synovial fibroblasts (SFBs) (n=6 each) were screened. DEG-associated co-expression and topological properties were analyzed to determine the rank of disease-associated genes. Specifically, the fold change of differentially expressed genes, the clustering coefficient and the degree of differential gene co-expression were integrated to determine the disease-associated gene ranking. The underlying molecular mechanisms of these crucial disease-associated genes were investigated by gene ontology (GO) enrichment analysis. A total of 1313 DEGs, including 1068 upregulated genes and 245 downregulated genes were observed. The top 20 disease-associated genes were identified, including proteoglycan 4, inhibin β B, carboxypeptidase M, alcohol dehydrogenase 1C and integrin β2. The major GO biological processes of these top 20 disease-associated genes were highly involved in the immune system, such as responses to stimuli, immune responses and inflammatory responses. This large-scale gene expression study observed disease-associated genes and their associated GO function in RA and OA, which may provide opportunities for biomarker development and novel insights into the molecular mechanisms of these two diseases.

Oxidative damage to mitochondrial DNA (mtDNA) and cell apoptosis are heavily implicated in aging. Our previous study established a mimetic rat model of aging in the cochleae using D-galactose (D-gal), and revealed that chronic injection of D-gal can increase oxidative stress and mtDNA common deletions (CD). The aim of the present study was to investigate the sources of reactive oxygen species and the occurrence of apoptosis in the cochleae of rats following 8 weeks of D-gal exposure. The results of the present study indicated that an elevated accumulation of the mtDNA CD and mitochondrial ultrastructural damage occurred in the cochleae of rats injected with D-gal for 8 weeks. In addition, the levels of 8-hydroxy-2-deoxyguanosine, NADPH oxidase (NOX) 3, P22phox and cleaved caspase 3, and the number of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end-labelling-positive cells were increased in the cochleae of D-gal-treated rats, compared with the controls. These findings suggested that nitric oxide synthase NOX3-associated oxidative stress may contribute to the accumulation of mtDNA mutations and activate a caspase 3-dependent apoptotic signalling pathway in the cochleae during aging. The present study also provided novel insights into the development of age-associated hearing loss, also termed presbycusis.