YTHDC1 inhibits osteoclast differentiation to alleviate osteoporosis by enhancing PTPN6 messenger RNA stability in an m6A-hUR-dependent manner.

YTHDC1 has been confirmed to mediate osteoporosis (OP) progression by regulating osteogenic differentiation. However, whether YTHDC1 mediates osteoclast differentiation and its molecular mechanism remains unclear. Quantitative real-time polymerase chain reaction and Western blot analysis were performed to detect the levels of YTHDC1, PTPN6, NFATc1, TRAP, RUNX2, alkaline phosphatase, and HUR. YTHDC1 knockout mice was constructed by CRISPR/Cas9 system, and the OP mice model was established by ovariectomy. Hematoxylin and eosin staining and micro-computed tomography were used to evaluate bone formation and bone mass. Mouse primary bone marrow macrophage cells were isolated and induced into osteoclasts. TRAP-positive cells were detected using TRAP staining. MeRIP-qPCR, RIP-qPCR assay, RNA affinity isolation assay, and co-immunoprecipitation assay were used to confirm the interactions among YTHDC1, PTPN6, and HUR. YTHDC1 expression was reduced and positively correlated with lumbar bone mineral density in OP patients. In the ovariectomy model of YTHDC1 knockout mice, bone formation was reduced, bone histomorphology was changed, and osteoclastic-related factor (NFATc1 and TRAP) levels were enhanced. Overexpression YTHDC1 inhibited osteoclast differentiation. YTHDC1 increased PTPN6 messenger RNA stability in an m6A-dependent manner. Moreover, YTHDC1 interacted with HUR to positively regulate PTPN6 expression. PTPN6 knockdown promoted osteoclast differentiation, and this effect was reversed by overexpressing HUR or YTHDC1. YTHDC1 was involved in regulating OP progression through inhibiting osteoclast differentiation by enhancing PTPN6 messenger RNA stability in an m6A-HUR-dependent manner.

Therapeutic effects of melatonin in female mice with central precocious puberty by regulating the hypothalamic Kiss-1/Kiss1R system.

In recent years, central precocious puberty (CPP) in children is becoming more common, which seriously affects their physical and psychological health and requires finding a safe and effective treatment method. The aim of this study was to investigate the therapeutic effect of melatonin on CPP. A CPP model was established by subcutaneous injection of 300 micrograms of danazol into 5-day-old female mice, followed by treatment with melatonin and leuprolide. The vaginal opening was checked daily. Mice were weighed, gonads were weighed, gonadal index was calculated, and gonadal development was observed by hematoxylin and eosin (HE) staining. Serum follicle stimulating hormone (FSH), luteinizing hormone (LH) and estradiol (E2) levels were measured by ELISA. By using RT-PCR and Western blotting, the mRNA and protein expression of the hypothalamus Kiss-1, Kiss-1 receptor (Kiss1R), gonadotropin-releasing hormone (GnRH), and pituitary GnRH receptor (GnRHR) were identified. The results showed that melatonin delayed vaginal opening time and reduced body weight, gonadal weight and indices in female CPP mice. Melatonin treatment prevents uterine wall thickening and ovarian luteinization in female CPP mice. Melatonin treatment reduces serum concentrations of FSH, LH, and E2 in female CPP mice. Melatonin suppressed the expressions of Kiss-1, Kiss1R and GnRH in the hypothalamus, and the expression of GnRHR in the pituitary of the female CPP mice. Our results suggest that melatonin can inhibit the hypothalamic-pituitary-gonadal (HPG) axis by down-regulating the Kiss-1/Kiss1R system, thereby treating CPP in female mice.

Construction and Chlorine Resistance of Thiophene-Poly(ethyleneimine)-Based Dual-Functional Nanofiltration Membranes.

The demand to improve the chlorine resistance of polyamide (PA) membranes is escalated with greater amounts of chlorine-containing disinfectant being used in global water treatment during the COVID-19 pandemic. In this work, we designed thiophene-functionalized poly(ethyleneimine) (TPEI) materials first and grafted them onto a conventional PA membrane to develop novel nanofiltration membranes (PEI-M, TPEI-1-M, TPEI-2-M). These membranes have dual-functionalized selective surfaces covered by hydrophilic amino groups and electron-rich thiophene moieties, which endow these membranes with superior chlorine resistance and improved separation performance. The modified membranes increase the rejection of MgCl2 from 86.5% of the nascent PA membrane (PA-M) to higher than 93.0% without sacrificing the membrane water permeability. More stable separation performance is achieved with all of the as-prepared membranes than PA-M after exposure to a 2000 ppm sodium hypochlorite solution. TPEI-2-M outperforms other membranes after being treated in a chlorination intensity of 16,000 ppm·h with the smallest flux loss and the highest MgCl2 rejection. This is mainly ascribed to the highest amount of amino and thiophene moieties on the TPEI-2-M surface. This study provides an effective protocol for developing novel PA-based nanofiltration membranes while demonstrating its superiority over current technologies with exceptional separation performance and antichlorine ability.

Effects of RFRP­3 on an ovariectomized estrogen­primed rat model and HEC­1A human endometrial carcinoma cells.

The hypothalamic peptide gonadotropin inhibitory hormone (GnIH) is a relatively novel hypothalamic neuropeptide, identified in 2000. It can influence the hypothalamic-pituitary-gonadal axis and reproductive function through various neuroendocrine systems. The present study aimed to explore the effects and potential underlying molecular mechanism of RFamide-related peptide-3 (RFRP-3) injection on the uterine fluid protein profile of ovariectomized estrogen-primed (OEP) rats using proteomics. In addition, the possible effects of RFRP-3 on the viability and apoptosis of the human endometrial cancer cell line HEC-1A and associated molecular mechanism were investigated. The OEP rat model was established through injection with GnIH/RFRP-3 through the lateral ventricle. At 6 h after injection, the protein components of uterine fluid of rats in the experimental and control groups were analyzed using liquid chromatography (LC)-tandem mass spectrometry (MS/MS). Differentially expressed proteins (DEPs) were analyzed using the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. Protein-protein interactions (PPI) were investigated using the STRING database. PPI networks were then established before hub proteins were selected using OmicsBean software. The expression of one of the hub proteins, Kras, was then detected using western blot analysis. Cell Counting Kit-8, Annexin V-FITC/PI, reverse transcription-quantitative PCR and western blotting were also performed to analyze cell viability and apoptosis. In total, 417 DEPs were obtained using LC-MS/MS, including 279 upregulated and 138 downregulated proteins. GO analysis revealed that the majority of the DEPs were secretory proteins. According to KEGG enrichment analysis, the DEPs found were generally involved in tumor-associated pathways. In particular, five hub proteins, namely G protein subunit α (Gna)13, Gnaq, Gnai3, Kras and MMP9, were obtained following PPI network analysis. Western blot analysis showed that expression of the hub protein Kras was downregulated following treatment with 10,000 ng/ml RFRP-3. RFRP-3 treatment (10,000 ng/ml) also suppressed HEC-1A cell viability, induced apoptosis, downregulated Bcl-2 and upregulated Bax protein expression, compared with those in the control group. In addition, compared with those in the control group, RFRP-3 significantly reduced the mRNA expression levels of PI3K, AKT and mTOR, while upregulating those of LC3-II. Compared with those in the control group, RFRP-3 significantly decreased the protein expression levels of PI3K, AKT, mTOR and p62, in addition to decreasing AKT phosphorylation. By contrast, RFRP-3 significantly increased the LC3-II/I ratio and G protein-coupled receptor 147 (GPR147) protein expression. In conclusion, the present data suggest that RFRP-3 can alter the protein expression profile of the uterine fluid of OEP rats by upregulating MMP9 expression whilst downregulating that of key hub proteins Gna13, GnaQ, Gnai3 and Kras. Furthermore, RFRP-3 can inhibit HEC-1A cell viability while promoting apoptosis. The underlying molecular mechanism may involve activation of GPR147 receptor by the direct binding of RFRP-3, which further downregulates the hub protein Kras to switch on the PI3K/AKT/mTOR pathway. This subsequently reduces the Bcl-2 expression and promotes Bax expression to induce autophagy.

Exogenous Melatonin Regulates Puberty and the Hypothalamic GnRH-GnIH System in Female Mice.

In recent years, the age of children entering puberty is getting lower and the incidence of central precocious puberty is increasing. It is known that melatonin plays an increasingly important role in regulating animal reproduction, but the specific role and mechanism of melatonin in regulating the initiation of puberty remain unclear. The purpose of the current study was to investigate the effect of subcutaneous melatonin injection on pubertal development in female mice and its mechanism of action. Female mice that were 22 days old received 1 mg/kg doses of melatonin subcutaneously every day for 10, 15 and 20 days. The vaginal opening was checked daily. Hematoxylin and eosin (HE) stain was used to determine the growth of the uterus and ovaries. Enzyme-linked immunosorbent assay (ELISA) was used to determine the levels of follicle-stimulating hormone (FSH), gonadotropin-inhibiting hormone (GnIH), and gonadotropin-releasing hormone (GnRH) in serum. By using RT-PCR and Western blotting, the mRNA and protein expression of the hypothalamus GnRH, GnIH, Kisspeptin (Kp), Proopiomelanocortin (POMC), Neuropeptide Y (NPY), as well as G protein-coupled receptor 147 (GPR147) were identified. The findings demonstrated that melatonin could suppress ovarian follicle and uterine wall growth as well as delay vaginal opening, decrease serum levels of GnRH and FSH and increase levels of GnIH. Melatonin increased GnIH and GPR147 expression in the hypothalamus in comparison to the saline group, while decreasing the expression of GnRH, Kisspeptin, POMC, and NPY. In conclusion, exogenous melatonin can inhibit the onset of puberty in female mice by modulating the expression of hypothalamic GnRH, GnIH, Kisspeptin, POMC and NPY neurons and suppressing the hypothalamic-pituitary-gonadal axis.

Direct effect of RFRP-3 microinjection into the lateral ventricle on the hypothalamic kisspeptin neurons in ovariectomized estrogen-primed rats.

RFamide-related peptide-3 (RFRP-3) may be involved in the inhibition of kisspeptin, but there is no direct evidence that RFRP-3 can directly act on kisspeptin neurons. The present study aimed to investigate the role and mechanism of RFRP-3 and kisspeptin in the hypothalamic-pituitary reproductive axis. In order to detect the expression and localization of RFRP-3 and kisspeptin in dorsomedial hypothalamic nucleus, double immunofluorescence method combined with confocal microscopy were performed. RFRP-3 was injected into the lateral ventricle of ovariectomized estrogen primed rats. Blood and brain tissues were collected at 60-, 120-, 240- and 360-min. Serum levels of gonadotropin-releasing hormone, luteinizing hormone and follicle-stimulating hormone were detected by ELISA. Kisspeptin expression in hypothalamus was detected by western blotting. Finally, surface plasmon resonance was used to verify whether RFRP-3 can directly interact with kisspeptin. Confocal images indicated that RFRP-3 and kisspeptin were co-expressed in the same neurons in the hypothalamus of ovariectomized estrogen-primed rats. Serum concentrations of gonadotropin-releasing hormone, luteinizing hormone and follicle-stimulating hormone were demonstrated to be significantly reduced following microinjection of RFRP-3 into the lateral ventricle for 60, 120, 240 and 360 min compared with the corresponding saline groups. The expression levels of kisspeptin in hypothalamus were gradually decreased following microinjection of RFRP-3 into the lateral ventricle. In addition, the affinity constant (KD) of RFRP-3 binding to kisspeptin was 6.005x10-5 M, indicating that RFRP-3 bound directly to kisspeptin in the range of protein-protein binding strength (KD, 10-3-10-6 M). In conclusion, RFRP-3 may regulate the hypothalamic-pituitary reproductive axis by inhibiting the expression of hypothalamic kisspeptin and direct binding.

Next-generation sequencing-based identification of EGFR and NOTCH2 complementary mutations in non-small cell lung cancer.

Although targeted therapy has emerged as an effective treatment strategy for non-small cell lung cancer (NSCLC), some patients cannot benefit from such therapy due to the limited number of therapeutic targets. The present study aimed to identify mutated genes associated with clinicopathological characteristics and prognosis and to screen for mutations that are not concurrent with applicable drug target sites in patients with NSCLC. Tumor tissue and blood samples were obtained from 97 patients with NSCLC. A lung cancer-specific panel of 55 genes was established and analyzed using next-generation sequencing (NGS). The results obtained from the clinical cohort were compared with the NSCLC dataset from The Cancer Genome Atlas (TCGA). Subsequently, 25 driver genes were identified by taking the intersection of the 55 lung-cancer-specific genes with three databases, namely, the Catalog of Somatic Mutations in Cancer database, the Network of Cancer Genes database and Vogelstein's list. Functional annotation and protein-protein interaction analysis were conducted on these 25 driver genes. The χ2 test and logistic regression were used to evaluate the association between mutations in the 25 driver genes and the clinicopathological characteristics of 97 patients, and phosphatase and tensin homolog (PTEN) and kirsten rat sarcoma viral oncogene homolog (KRAS) were associated with stage at diagnosis and sex, respectively, while epidermal growth factor receptor (EGFR) was associated with sex, stage at diagnosis, metastasis, CEA and CYFRA21-1. Moreover, the association between the 25 driver gene mutations and overall survival were examined using Cox regression analysis. Age and Notch homolog 2 (NOTCH2) mutations were independent prognostic factors in TCGA dataset. The correlations between statistically significant mutations in EGFR, KRAS, PTEN and NOTCH2 were further examined, both in the clinical data and TCGA dataset. There was a negative correlation between EGFR and NOTCH2 mutations (correlation coefficient, -0.078; P=0.027). Thus, the present study highlights the importance of NOTCH2 mutations and might provide novel therapeutic options for patients with NSCLC who do not harbor EGFR mutations.

Overexpression of circ_0021739 in Peripheral Blood Mononuclear Cells in Women with Postmenopausal Osteoporosis Is Associated with Reduced Expression of microRNA-194-5p in Osteoclasts.

BACKGROUND Postmenopausal osteoporosis, a common disease among elderly women, is linked to estrogen deficiency, mechanical loading, and genotype. Circular RNAs (circRNAs) are formed through reverse splicing of the splice donor at the 3' end and the splice accepter at the 5' end in pre-mRNA and have been shown to be involved in the development of multiple diseases. Based on their high sequence conservation and stability, circRNAs may be useful biomarkers in different diseases. However, the roles of circRNAs in postmenopausal osteoporosis remain incompletely understood. MATERIAL AND METHODS Fifty-three postmenopausal women were assigned to either the postmenopausal osteoporosis group (n=28) or the control group (n=25). Reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR) analysis was performed to determine the differential expression of circRNAs between the 2 groups. Receiver-operating characteristic (ROC) curve analysis was conducted to evaluate the clinical diagnostic value of circRNA. Prediction of the binding sites between circRNA and miRNAs was conducted using miRanda and RNAhybrid. The function of the circRNA in osteoclastogenesis was determined by circRNA overexpression followed by tartrate-resistant acid phosphatase staining and RT-qPCR analysis. RESULTS Among 4 circRNAs previously identified by RNA-sequencing analysis as differentially expressed in patients with postmenopausal osteoporosis, only hsa_circ_0021739 showed a significant difference in expression between the groups and was downregulated in patients with postmenopausal osteoporosis. The hsa_circ_0021739 expression level was determined to be correlated with the lumbar vertebra, femur, and forearm T-scores. Overexpression of hsa_circ_0021739 decreased the level of hsa-miR-502-5p and inhibited the differentiation of osteoclasts. CONCLUSIONS The circRNA hsa_circ_0021739 is a potential blood biomarker for postmenopausal osteoporosis. In addition, hsa-miR-502-5p is a likely target of hsa_circ_0021739, which acts to regulate the differentiation of osteoclasts.

Moringa oleifera leaf extracts protect BMSC osteogenic induction following peroxidative damage by activating the PI3K/Akt/Foxo1 pathway.

OBJECTIVE: We aimed to investigate the therapeutic effects of Moringa oleifera leaf extracts on osteogenic induction of rat bone marrow mesenchymal stem cells (BMSCs) following peroxidative damage and to explore the underlying mechanisms. METHODS: Conditioned medium was used to induce osteogenic differentiation of BMSCs, which were treated with H2O2, Moringa oleifera leaf extracts-containing serum, or the phosphatidyl inositol-3 kinase (PI3K) inhibitor wortmannin, alone or in combination. Cell viability was measured using the MTT assay. Cell cycle was assayed using flow cytometry. Expression levels of Akt, phosphorylated (p)Akt, Foxo1, and cleaved caspase-3 were analyzed using western blot analysis. The mRNA levels of osteogenesis-associated genes, including alkaline phosphatase (ALP), collagen І, osteopontin (OPN), and Runx2, were detected using qRT-PCR. Reactive oxygen species (ROS) and malondialdehyde (MDA) levels, as well as superoxide dismutase (SOD), glutathione peroxidase (GSH-PX), and ALP activity were detected using commercially available kits. Osteogenic differentiation capability was determined using alizarin red staining. RESULTS: During osteogenic induction of rat BMSCs, H2O2 reduced cell viability and proliferation, inhibited osteogenesis, increased ROS and MDA levels, and decreased SOD and GSH-PX activity. H2O2 significantly reduced pAkt and Foxo1 expression, and increased cleaved caspase-3 levels in BMSCs. Additional treatments with Moringa oleifera leaf extracts partially reversed the H2O2-induced changes. Wortmannin partially attenuated the effects of Moringa oleifera leaf extracts on protein expression of Foxo1, pAkt, and cleaved caspase-3, as well as mRNA levels of osteogenesis-associated genes. CONCLUSION: Moringa oleifera leaf extracts ameliorate peroxidative damage and enhance osteogenic induction of rat BMSCs by activating the PI3K/Akt/Foxo1 pathway.

Sericin inhibits MDA­MB­468 cell proliferation via the PI3K/Akt pathway in triple­negative breast cancer.

Triple negative breast cancer (TNBC) is a subtype of breast cancer characterized by an aggressive histology and poor prognosis, with limited treatment options in the clinic. In the present study, the effect of sericin, as an anti­cancer drug, on TNBC cell proliferation was investigated using a MTT assay, a colony formation assay and immunocytochemistry staining of Ki67. Results from the flow cytometry demonstrated that sericin induced G0/G1 cell cycle arrest and promoted cellular apoptosis. Cell cycle and apoptosis­related proteins were detected via western blot analysis. Immunocytochemistry staining identified that P21 was translocated into the nucleus. Additionally, several pathways were significantly enriched in TNBC based on the Gene Expression Omnibus database, with the most prominent pathway being the PI3K/Akt signaling pathway. In TNBC MDA­MB­468 cells, sericin suppressed the PI3K/Akt pathway. All these findings suggested that sericin served a critical role in suppressing TNBC cell proliferation, inducing cell cycle arrest and promoting cellular apoptosis. The results indicated that the underlying molecular mechanism was, at least partially, via the downregulation of the PI3K/Akt signaling pathway.

Characterization of Dysregulated lncRNA-Associated ceRNA Network Reveals Novel lncRNAs With ceRNA Activity as Epigenetic Diagnostic Biomarkers for Osteoporosis Risk.

The altered expression of long non-coding RNAs (lncRNAs) has been implicated in the development and human diseases. However, functional roles and regulatory mechanisms of lncRNA as competing endogenous RNAs (ceRNAs) in osteoporosis and their potential clinical implication for osteoporosis risk are largely unexplored. In this study, we performed integrated analysis for paired expression profiles and regulatory relationships of dysregulated lncRNAs, mRNAs, and miRNAs based on "ceRNA hypothesis," and constructed an osteoporosis-related dysregulated miRNA-mediated lncRNA-mRNA ceRNA network (DysCeNet) composed of 105 nodes (including eight miRNAs, 24 mRNAs, and 73 lncRNAs) and 515 edges. Functional analysis suggested that the DysCeNet was involved in known osteoporosis or bone metabolism-related biological processes and pathways. Then, we performed random forest-based feature selection for 73 lncRNAs with ceRNA activity and identified 25 of 73 lncRNAs as potential diagnostic biomarkers. A random forest-based classifier composed of 25 lncRNA biomarkers (RF-25lncRNA) was developed for predicting osteoporosis risk. Performance evaluation with the leave-one-out cross-validation (LOOCV) procedure showed that the RF-25lncRNA achieved a good performance in distinguishing high- and low-bone mineral density (BMD) subjects in different osteoporosis datasets. Our study for the first time revealed a global view of lncRNA-associated ceRNA regulation in osteoporosis and provided novel lncRNAs with ceRNA activity as candidate epigenetic diagnostic biomarkers for early detection of osteoporosis risk.

Thioredoxin mitigates H2 O2 -induced inhibition of myogenic differentiation of rat bone marrow mesenchymal stem cells by enhancing AKT activation.

Thioredoxin (Trx) is a hydrogen acceptor of ribonucleotide reductase and a regulator of some enzymes and receptors. It has been previously shown that significantly elevated levels of Trx expression are associated with the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs), but it is not clear how Trx regulates the effects of hydrogen peroxide (H2 O2 ) on myogenic differentiation of BMSCs. Here, we report that rat BMSCs treated with a high dose (150 µm) of H2 O2 exhibited a significant reduction in viability, cell cycling, and superoxide dismutase and glutathione peroxidase levels, and an increase in reactive oxygen species and malondialdehyde levels, which was accompanied by reductions in protein kinase B activation and forkhead Box O1, myogenic differentiation 1 and myogenin expression during myogenic differentiation. Furthermore, treatment with recombinant human Trx significantly mitigated the effects of H2 O2 on the myogenic differentiation of BMSCs, and this was abrogated by cotreatment with wortmannin [a specific phosphatidylinositol 3-kinase inhibitor]. In summary, our results suggest that treatment with recombinant human Trx mitigates H2 O2 -induced oxidative stress and may promote myogenic differentiation of rat BMSCs by enhancing phosphatidylinositol 3-kinase/protein kinase B/forkhead Box O1 signaling.

Sericin enhances the insulin-PI3K/AKT signaling pathway in the liver of a type 2 diabetes rat model.

The aim of the current study was to investigate the regulatory effect of sericin on the hepatic insulin-phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway in a type 2 diabetes rat model. Male Sprague Dawley rats were randomly divided into four groups: Control group, diabetic model group, high-dose sericin group and low-dose sericin group, with 12 rats in each group. Fasting blood glucose was detected by the glucose oxidase method, and hepatic glycogen was determined by periodic acid-Schiff staining. The morphology of the liver was observed by hematoxylin and eosin staining. Immunohistochemical staining, western blotting and reverse transcription-quantitative polymerase chain reaction were used to determine the protein and mRNA expression levels of insulin receptor (IR), IR substrate-1 (IRS-1), PI3K and AKT. Compared with the control group, the blood glucose of the diabetic model group was significantly increased (P<0.05). The glycogen content and the expression levels of IR, IRS-1, PI3K and AKT in the diabetic model group were significantly lower (P<0.05), and the liver morphological structure of the diabetic model group exhibited obvious pathological changes compared with the control group. Compared with the diabetic model group, the blood glucose of the high- and low-dose sericin groups was significantly reduced, while the glycogen content and the expression levels of IR, IRS-1, PI3K and AKT in the sericin treatment groups were significantly increased (P<0.05). Additionally, the liver pathological changes of high-dose and low-dose sericin groups were markedly reduced. Sericin may enhance the signaling transduction effect of insulin by upregulating the expression levels of key factors (IR, IRS-1, PI3K and AKT) in the liver insulin-PI3K/AKT signaling pathway, thus promoting glucose transport and liver glycogen synthesis, and further reducing blood glucose.

TRIM28 knockdown increases sensitivity to etoposide by upregulating E2F1 in non-small cell lung cancer.

Tripartite motif containing 28 (TRIM28) is a universal corepressor for Kruppel­associated box zinc finger proteins. In our previous study, it was shown that expression of TRIM28 is upregulated in non­small cell lung cancer (NSCLC) cell lines and tissues. Here, we demonstrated that the stable silencing of TRIM28 expression by a specific siRNA lentivirus vector increased the sensitivity of NSCLC cells to chemotherapeutic agent etoposide. Combination of TRIM28 siRNA and etoposide significantly inhibited the growth and proliferation of lung adenocarcinoma PAa cells and exerted obvious antitumor effects in nude mice. Using FCM and terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) assay, we found that TRIM28 siRNA in combination with etoposide increased apoptosis in vitro and in vivo which was induced by E2F1 activity, since the expression of E2F1 and its target genes was significantly increased in the cotreatment group. Cell proliferation and apoptosis were almost completely abolished in the PAa cells cotreated with TRIM28 siRNA and etoposide following knockdown of E2F1. The results of our study demonstrated that the combination of TRIM28 siRNA and etoposide may be effective against NSCLC and has the potential of being a new therapeutic tool for future treatment.

Antitumour activity of 2-dihydroailanthone from the bark of Ailanthus altissima against U251.

Context The bark of Ailanthus altissima (Mill.) Swingle (Simaroubaceae) is traditionally used to treat ascariasis, diarrhoea, spermatorrhoea, bleeding and gastrointestinal diseases. Objective The objective of this study is to investigate the antitumour activity and mechanism of 2-dihydroailanthone isolated from A. altissima. Materials and methods The U251 cells were treated with 1.00, 4.00 and 8.00 µg/mL of 2-dihydroailanthone for 48 h and the normal cells treated with 20.00 µg/mL of 2-dihydroailanthone were tested as well. Proliferation inhibition of 2-dihydroailanthone on the cells was tested by MTT. Apoptosis and cell-cycle distribution in U251 cells with 1.00, 3.00 and 5.80 µg/mL of 2-dihydroailanthone for 48 h were determined by flow cytometry, respectively. The expression of the apoptosis-related genes and proteins was analysed by RT-PCR and Western blot method, respectively. Results MTT assay revealed that 2-dihydroailanthone inhibited U251 cells proliferation. The cell viability of U251 cells was 62.82, 31.34 and 25.58%, and that of three normal cells was 72.75, 82.74 and 44.92%, respectively. Flow cytometry assay showed that 2-dihydroailanthone induced apoptosis and G0/G1 phase cycle arrest towards U251 cells. The late apoptotic cells were 11.37, 21.73 and 33.83%, and the cells cycle distributed in the G0/G1 accounted for 48.85, 62.77 and 64.40%, respectively. The Western blot and RT-PCR assay showed that up-regulation of pro-apoptotic bax protein and down-regulation of anti-apoptotic bcl-2 protein as well as their mRNA on U251 cells might be related to the apoptosis induction and proliferation inhibition. Conclusion An important bioactive component, 2-dihydroailanthone, has antitumour effects, enlightening a novel source of phytomedicines in tumour therapy.

[LC-MS/MS Analysis on the Components of Excretory-secretory Protein of Trichinella spiralis Muscle Larvae].

Objective: To analyze the components of excretory-secretory protein（ESP） of Trichinella spiralis muscle larvae, and search for the anti-tumor protein components. Methods: The Trichinella spiralis muscle larvae were collected, and ESP was prepared. The ESP was separated in 15% SDS-PAGE. Proteins extracted from the protein bands were lysed with trypsin, and analyzed by LC-MS/MS. The identified proteins were classified by Gene Ontology（GO） according to cell component, molecular function, and biological processes. Results: SDS-PAGE revealed clear protein bands at Mr 10 000-142 000. A total of 162 proteins were analyzed with LC-MS/MS, of which 63 were identified, 34 were putative proteins, and 65 were unidentified proteins. Six anti-tumor relevant proteins were revealed, which were tropomyosin, histone H2A, cleavage and polyadenylation specificity factor subunit 2, serine proteinase inhibitor Kazal-type 4, Armadillo segment polarity protein and eukaryotic initiation factor 4A. The GO enrichment analysis showed that the identified proteins possessed 54 different types of molecular functions, and participated in cell structure and 382 biological processes. Conclusion: The ESP of Trichinella spiralis muscle larvae has complex protein components, many with unknown identities. Six anti-tumor relevant proteins were determined from the 63 identified proteins.

NPY1R is a novel peripheral blood marker predictive of metastasis and prognosis in breast cancer patients.

The aim of the current study was to evaluate a novel tumor marker, neuropeptide Y receptor Y1 (NPY1R), for the detection of circulating cancer cells and to investigate its clinical significance in breast cancer patients. The Digital Gene Expression Displayer tool of the Cancer Genome Anatomy Project was used to identify the marker gene NPY1R, which is able to detect circulating cancer cells. Nested quantitative polymerase chain reaction was performed to correlate the NPY1R expression levels with the clinicopathological features of 142 breast cancer patients. A follow-up study of 131 of the breast cancer patients was conducted for 38 months. Compared with the 60 normal control individuals, NPY1R was highly expressed in the cancer patients (P<0.01). These high levels of NPY1R expression were positively correlated with the clinical stage and lymph node metastasis status of the disease, as well as with the status of the estrogen and progesterone receptors (P<0.05). Breast cancer patients with circulating cancer cells that expressed NPY1R exhibited shorter tumor-specific survival when compared with those with no NPY1R expression (P<0.01). Additionally, the mortality rate was associated with HER2 expression in the NPY1R positive and negative groups. These results indicate that NPY1R may serve as a useful marker to predict breast cancer metastasis and to evaluate the prognosis of breast cancer patients.

A rapid nested polymerase chain reaction method to detect circulating cancer cells in breast cancer patients using multiple marker genes.

The aim of the present study was to develop a simple and rapid method for the detection of circulating cancer cells using multiple tumor markers and to investigate the clinical significance of circulating cancer cells in breast cancer patients. A novel rapid nested polymerase chain reaction (PCR) assay, with high sensitivity and specificity, was evaluated, which was considered to be suitable for clinical application. The rapid nested PCR method was used to detect the circulating cancer cells of 142 breast cancer patients, using a panel of marker genes (FAM83A, NPY1R and KRT19), which were identified by the Digital Gene Expression Displayer Tool of the National Cancer Institute-Cancer Genome Anatomy Project. In total, 79.6% of the 142 breast cancer patient blood samples were found to express at least one tumor marker. In addition, the number of positive markers was found to significantly correlate with the disease stage and presence of distant metastasis. Furthermore, positivity for more than one tumor marker appeared to predict a reduced survival time in breast cancer patients.

The relationship between seven common polymorphisms from five DNA repair genes and the risk for breast cancer in northern Chinese women.

BACKGROUND: Converging evidence supports the central role of DNA damage in progression to breast cancer. We therefore in this study aimed to assess the potential interactions of seven common polymorphisms from five DNA repair genes (XRCC1, XRCC2, XRCC3, XPA and APEX1) in association with breast cancer among Han Chinese women. METHODOLOGY/PRINCIPAL FINDINGS: This was a case-control study involving 606 patients diagnosed with sporadic breast cancer and 633 age- and ethnicity-matched cancer-free controls. The polymerase chain reaction-ligase detection reaction method was used to determine genotypes. All seven polymorphisms were in accordance with Hardy-Weinberg equilibrium in controls. Differences in the genotypes and alleles of XRCC1 gene rs25487 and XPA gene rs1800975 were statistically significant between patients and controls, even after the Bonferroni correction (P<0.05/7). Accordingly, the risk for breast cancer was remarkably increased for rs25487 (OR = 1.28; 95% CI: 1.07-1.51; P = 0.006), but decreased for rs1800975 (OR = 0.77; 95% CI: 0.67-0.90; P = 0.001) under an additive model at a Bonferroni corrected alpha of 0.05/7. Allele combination analysis showed higher frequencies of the most common combination C-G-G-C-G-G-G (alleles in order of rs1799782, rs25487, rs3218536, rs861539, rs1800975, rs1760944 and rs1130409) in controls than in patients (PSim = 0.002). In further interaction analysis, two-locus model including rs1800975 and rs25487 was deemed as the overall best model with the maximal testing accuracy of 0.654 and the cross-validation consistency of 10 out of 10 (P = 0.001). CONCLUSION: Our findings provide clear evidence that XRCC1 gene rs25487 and XPA gene rs1800975 might exert both independent and interactive effects on the development of breast cancer among northern Chinese women.

A comparison study between CT angiography with 64-multislice spiral computed tomography and selective X-ray coronary angiography.

The aim of this study was to evaluate the diagnostic value of 64-multislice spiral computed tomography (64-MSCT) for coronary stenosis compared with selective X-ray coronary angiography (SCA). Patients with chest pain, chest tightness or coronary stenosis received SCA and they acted as the controls. The sensitivity and accuracy of 64-MSCT were analyzed as compared with SCA. Images from 64-MSCT were obtained for 95 patients. For the diagnosis of myocardial bridge, 64-MSCT coronary CT angiography (CTA) is superior to SCA. In cases of mild coronary stenosis, combined with clinical symptoms, patients may choose to receive conservative treatment instead of SCA. However, cases of moderate coronary stenosis should receive SCA to determine the diagnosis. In conclusion, no difference was observed between 64-MSCT coronary CTA and SCA in their ability to exclude true negative diagnoses and diagnosing true positives of coronary disease. The 64-MSCT coronary CTA method produces improved image quality for diagnosis of coronary stenosis and is a non-invasive, reliable and effective method for the diagnosis of the severity of coronary stenosis.