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Lung cancer is one familiar cancer that threatens the lives of humans. circCTNNB1 has been disclosed to have regulatory functions in some diseases. However, the functions and related regulatory mechanisms of circCTNNB1 in lung cancer remain largely indistinct. The mRNA and protein expression levels were examined through real-time polymerase chain reaction (RT-qPCR) and western blot. The cell proliferation was tested through CCK-8 assay. The cell migration and invasion were confirmed through Transwell assays. The cell senescence was evaluated through SA-ß-gal assay. The binding ability between miR-186-5p and circCTNNB1 (or YY1) was verified through luciferase reporter and RIP assays. In this study, the higher expression of circCTNNB1 was discovered in lung cancer tissues and cell lines and resulted in poor prognosis. In addition, circCTNNB1 facilitated lung cancer cell proliferation, migration, invasion, and suppressed cell senescence. Knockdown of circCTNNB1 retarded the Wnt pathway. Mechanism-related experiments revealed that circCTNNB1 combined with miR-186-5p to target YY1. Through rescue assays, YY1 overexpression could rescue decreased cell proliferation, migration, invasion, increased cell senescence, and retarded Wnt pathway mediated by circCTNNB1 suppression. Furthermore, YY1 acts as a transcription factor that can transcriptionally activate circCTNNB1 to form YY1/circCTNNB1/miR-186-5p/YY1 positive loop. Through in vivo assays, circCTNNB1 accelerated tumour growth in vivo. All findings revealed that a positive loop YY1/circCTNNB1/miR-186-5p/YY1 aggravated lung cancer progression by modulating the Wnt pathway.

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PURPOSE: Atherosclerosis causes plaque to build-up in arteries. Effect of the specific local hemodynamic environment around an atherosclerotic plaque on the thrombosis formation does not remain quite clear but is believed to be crucial. The aim of this study is to uncover the flow effects on plaques formation. METHODS: To study the mechanically regulated plaque formation, the flow fields in artery blood vessels with different stenosis rates at various Reynolds numbers were simulated numerically with the two-dimensional axisymmetric models, and the hemodynamic characteristics around the plaque were scaled with stenosis rate and Reynolds number. RESULTS: The results showed that increases of both Reynolds number and stenosis rate facilitated the occurrence of flow separation phenomenon, extended recirculation zone, and upregulated the maximum normalized wall shear stress near the plaque throat section while downregulated the minimal normalized wall shear stress at the front shoulder of plaque, as it should be; in the atherosclerotic plaque leeside of the recirculation zone, an obvious catch bond region of wall shear stress might exist especially under low Reynolds number with stenosis rate smaller than 30%. This catch bond region in the plaque leeside might be responsible for the LBF (low blood flow)-enhanced formation of the atherosclerotic plaque. CONCLUSIONS: This work may provide a novel insight into understanding the biomechanical effects behind the formation and damage of atherosclerotic plaques and propose a new strategy for preventing atherosclerotic diseases.

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OBJECTIVE: Glycogen phosphorylase B (PYGB), the rate-determining enzyme in glycogen degradation, plays a critical role in progression of various tumors. The present study focused on the potential molecular mechanism toward PYGB in non-small cell lung cancer (NSCLC) progression. METHODS: Expression of PYGB in NSCLC tissues and cell lines was evaluated via quantitative real-time PCR (qRT-PCR), western blot and immunohistochemistry. Cell viability, proliferation and apoptosis were investigated using 3-(4,5-Dimethylthiazol)-2,5-diphenyltetrazolium bromide (MTT) assay, 5-bromo-2-deoxyuridine (BrdU) and flow cytometry, respectively. Cell migration and invasion ability were detected by wound healing and transwell invasion assays, respectively. The in vivo effect of PYGB on NSCLC tumor growth was determined via subcutaneous xenotransplanted tumor model. RESULTS: PYGB was upregulated in NSCLC tissues and cell lines, suggesting a poor prognosis in NSCLC patients. In vitro functional assays indicated that knockdown of PYGB suppressed cell viability, proliferation, migration and invasion, while promoted cell apoptosis in NSCLC. Mechanistically, we found that overexpression of PYGB could activate phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway, while these effects were effectively reversed by knockdown of PYGB. In vivo tumorigenesis and PI3K/AKT signaling pathway were also inhibited by PYGB knockdown. CONCLUSIONS: Knockdown of PYGB suppressed NSCLC progression, suggesting PYGB as a novel biomarker and potential molecular therapeutic target for further investigation in NSCLC.

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Tyrosine kinase inhibitors (TKIs) bring significant benefits for patients with cancers harboring epidermal growth factor receptor (EGFR) mutations. However, after treatment for a certain period, most patients ultimately acquire resistance. Numerous studies indicated that PI3K has an important role in tumor cell growth and drug sensitivity. Furthermore, inhibition of PI3K may lead to sensitization of non-small cell lung cancer (NSCLC) cells to EGFR-TKIs. The aim of the present study was to explore whether LY294002, an inhibitor of PI3K, is able to improve the sensitivity of NSCLC cell lines with wild-type EGFR to the EGFR-TKI erlotinib. An MTT assay was used to examine the effect of combined treatment with LY294002 and erlotinib on cell survival of two EGFR wild-type NSCLC cell lines, NCI-H661 and NCI-H460. Furthermore, flow cytometry was used to assess apoptosis in NCI-H661 and NCI-H460 cells after treatment with erlotinib and LY294002. In addition, the expression of downstream proteins was detected by western blot analysis. The results indicated that the number of viable NCI-H661 and NCI-H460 cells was dose-dependently reduced by erlotinib or LY294002. Compared to treatment with erlotinib alone, the cell apoptosis was enhanced if combined treatment of erlotinib and LY294002 was performed in NCI-H661 cells. Furthermore, combination treatment of erlotinib and LY294002 resulted in a significant reduction of phosphorylated p70S6K levels in NCI-H661 [PI3K catalytic subunit alpha (PI3KCA) wild-type] cells. However, this phenomenon was not observed in the NCI-H460 cell line (PIK3CA mutant-type). In conclusion, the present study indicated that inhibition of PI3K may have the potential to improve the sensitivity of NSCLC cells to an EGFR-TKI. However, the therapeutic effect may depend on the mutation status of PIK3CA.

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BACKGROUND: Long non-coding RNA (lncRNA) HOTAIR has been reported to be associated with cisplatin (DDP) resistance in different human cancers including non-small cell lung cancer (NSCLC). However, the mechanism of HOTAIR in cisplatin resistance of NSCLC remains largely undefined. MATERIALS AND METHODS: Expression of HOTAIR, miR-149-5p and doublecortin-like kinase 1 (DCLK1) was detected using real-time quantitative PCR (RT-qPCR) and Western blotting. Cisplatin resistance was determined with cell counting kit (CCK)-8 assay and transwell assays in vitro, and xenograft tumor models in vivo. The target binding between miR-149-5p and either HOTAIR or DCLK1 was predicted on Diana Tools website, and confirmed by dual-luciferase reporter assay and RNA immunoprecipitation. RESULTS: Expression of HOTAIR was upregulated in DDP-resistant NSCLC tumor tissues and cell lines (A549/DDP and H1299/DDP). Knockdown of HOTAIR decreased the acquired cisplatin resistance of A549/DDP and H1299/DDP cells, as evidenced by attenuated 50% inhibitory concentration (IC50) of DDP, cell proliferation, migration and invasion in vitro, as well as tumor growth inhibition in vivo. Mechanically, HOTAIR negatively regulated miR-149-5p expression via targeting, and DCLK1 was a downstream target for miR-149-5p. DCLK1 was indirectly regulated by HOTAIR in DDP-resistant NSCLC cells as well. Functionally, miR-149-5p deletion could counteract the inhibitory effect of HOTAIR knockdown on cisplatin resistance; contrarily, restoring miR-149-5p exhibited the similar inhibition on cisplatin resistance in DDP-resistant cells in vitro, which was then abated by DCLK1 upregulation. CONCLUSION: Knockdown of HOTAIR enhances DDP-resistant NSCLC cells to overcome cisplatin resistance partially via regulating miR-149-5p/DCLK1 axis.

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The destruction of proteins via the ubiquitin-proteasome system is a multi-step, complex process involving polyubiquitination of substrate proteins, followed by proteolytic degradation by the macromolecular 26S proteasome complex. Inhibitors of the proteasome promote the accumulation of proteins that are deleterious to cell survival and are promising anticancer agents. Oprozomib (OPZ), an oral second-generation proteasome inhibitor, has been shown to inhibit the growth of several cancers in preclinical and clinical trials, including multiple myeloma and head and neck cancers, but its effects on lung cancer has not yet been determined. In this study, we evaluated the inhibitory effects of OPZ on lung cancer cell lines in vitro. The results showed that OPZ significantly suppressed cell proliferation and strongly induced apoptosis in both tested lung cancer cells independent of p53 expression. OPZ was able to cause obvious caspase 3 and PARP cleavages and stabilize p53 and its transcriptional targets p21, PUMA, and Noxa. Moreover, OPZ was capable of sensitizing lung cancer cells to the conventional chemotherapeutic drug cisplatin. Our study provides preclinical data and sheds light on the potential applications of proteasome inhibitor OPZ in lung cancer treatment.

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The inhibition of cholinesterases (ChEs) by carbamates includes a carbamylation (inhibition) step, in which the drug transfers its carbamate moiety to the active site of the enzyme and a decarbamylation (activity recovery) step, in which the carbamyl group is hydrolyzed from the enzyme. The carbamylation and decarbamylation kinetics decide the extent and the duration of the inhibition, thus the full characterization of candidate carbamate inhibitors requires the measurement of the kinetic constants describing both steps. Carbamylation and decarbamylation rate constants are traditionally measured by two separate set of experiments, thus making the full characterization of candidate inhibitors time-consuming. In this communication we show that by the analysis of the area under the inhibition-time curve of cholinesterases inhibited by carbamates it is possible to calculate the decarbamylation rate constant from the same data traditionally used to characterize only the carbamylation kinetics, therefore it is possible to obtain a full characterization of the inhibition with a single set of experiments. The characterization of the inhibition kinetics of human and dog plasma butyrylcholinesterase and of human acetylcholinesterase by bambuterol and bambuterol monocarbamate enantiomers was used to demonstrate the validity of the approach. The results showed that the proposed method provides reliable estimations of carbamylation and decarbamylation rate constants thus representing a simple and useful approach to reduce the time required for the characterization of carbamate inhibitors.

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Several molecules containing carbamate groups are metabolized by cholinesterases. This metabolism includes a time-dependent catalytic step which temporary inhibits the enzymes. In this paper we demonstrate that the analysis of the area under the inhibition versus time curve (AUIC) can be used to obtain a quantitative estimation of the amount of carbamate metabolized by the enzyme. (R)-bambuterol monocarbamate and plasma butyrylcholinesterase were used as model carbamate-cholinesterase system. The inhibition of different concentrations of the enzyme was monitored for 5h upon incubation with different concentrations of carbamate and the resulting AUICs were analyzed. The amount of carbamate metabolized could be estimated with <15% accuracy (RE%) and ≤23% precision (RSD%). Since the knowledge of the inhibition kinetics is not required for the analysis, this approach could be used to determine the amount of drug metabolized by cholinesterases in a selected compartment in which the cholinesterase is confined (e.g. in vitro solutions, tissues or body fluids), either in vitro or in vivo.

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The present study investigated the correlation and significance of HOX transcript antisense RNA (HOTAIR) and epithelial-mesenchymal transition (EMT)-related factors in the occurrence and metastasis of esophageal squamous cell cancer (ESCC) progression. The mRNA and protein expression levels of HOTAIR and EMT­related factors were detected in 96 ESCC and para­carcinoma tissues using reverse transcription­quantitative polymerase chain reaction and western blot analysis. The expression levels of these factors, and the correlation between these factors and clinicopathological characteristics were subsequently analyzed. HOTAIR mRNA expression levels were significantly higher in ESCC compared with in para-carcinoma tissues, and HOTAIR mRNA expression levels were significantly higher in the groups with lymph node involvement or organ metastasis compared with the group without. Furthermore, HOTAIR expression levels demonstrated a significant increasing trend from well­differentiated cancer to poorly differentiated cancer. The mRNA and protein expression levels of zinc finger protein SNAI1 (Snail) and ß­catenin in ESCC were significantly higher compared with para-carcinoma tissues, whereas E­cadherin mRNA and protein expression levels were lower in ESCC tissues compared with in para-carcinoma tissues. Snail mRNA and protein expression levels were also significantly higher in groups with lymph node involvement or organ metastasis compared with those without, and ß­catenin protein expression levels were significantly higher in the groups with lymph node involvement or organ metastasis compared with the group without. In the 96 ESCC tissues, HOTAIR mRNA expression levels were positively correlated with Snail mRNA and protein expression levels, and were negatively correlated with E­cadherin expression levels. HOTAIR mRNA expression levels were also positively correlated with ß­catenin mRNA expression levels. In conclusion, HOTAIR may be involved in carcinogenesis and metastasis, and may induce the expression of EMT­related factors; detection of these factors may assist in early diagnosis and prognostic prediction.

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Epithelial-mesenchymal transition (EMT) is a critical cellular process in cancer metastasis, during which epithelial polarized cells become motile mesenchymal cells. Since transforming growth factor-ß (TGF-ß) is a potent inducer of EMT, blocking of TGF-ß/Smad signaling has become a promising cancer therapy. Nobiletin, a polymethoxy flavonoid from Citrus depressa, has been shown to be valuable for cancer treatment, yet the mechanism remains unclear. In the present study, lung adenocarcinoma A549 and H1299 cells were used to evaluate the effect of nobiletin on EMT induced by TGF-ß1. Nobiletin successfully inhibited TGF-ß1-induced EMT, migration, invasion and adhesion in vitro, accompanied by attenuation of MMP-2, MMP-9, p-Src, p-FAK, p-paxillin, Snail, Slug, Twist and ZEB1 expression. Nobiletin inhibited the transcriptional activity of Smads without changing the phosphorylation status or translocation of Smads induced by TGF-ß1. Moreover, Smad3 is requisite in TGF-ß1-stimulated EMT. Smad3 overexpression meaningfully impaired the ability of nobiletin to reverse TGF-ß1-induced EMT. In vivo, nobiletin prohibited the growth of metastatic nodules in the lungs of nude mice. Moreover, nobiletin inhibited tumor growth and reversed EMT in mice bearing A549-Luc xenografts, as revealed by IVIS imaging and immunohistochemical analysis. Collectively, the data suggest that nobiletin prevents EMT by inactivating TGF-ß1/Smad3 signaling.

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BACKGROUND: This study was designed to investigate epidermal growth factor receptor (EGFR) mutation types affecting lung cancer treatment in patients in Xinjiang, China. We detected and analyzed differences in the EGFR mutation points of Uighur and Han patients with lung adenocarcinoma. We examined 181 specimens of lung adenocarcinoma tissue embedded with paraffin (76 Uighur and 105 Han patients) for mutations in the EGFR gene in exon 18-21 by the amplification refractory mutation system (ARMS) method. We used the chi-square statistical method to analyze the relationship between mutations and patients' clinical parameters. RESULTS: EGFR somatic mutations were detected in 59 of 181 cases (32.6%). The mutation rate was higher in Han patients (45.7%) than in Uighur patients (15.8%) (P < 0.001). The main mutation types were the exon 19 deletion and the L858R point mutation in exon 21. In Han patients we found 21 (44.7%) cases of exon 19 deletion, 24 (51.1%) cases of L858R in exon 21, 1 case (2.1%) with mutations in both exon 19 and exon 21, and 1 case (2.1%) with T790 mutation in exon 20. In Uighur patients we found 8 (66.7%) cases of exon 19 deletion and 4 (33.3%) cases of L858R in exon 21. CONCLUSIONS: In comparing these groups, the exon 19 deletion was more common than L858R in exon 21 in Uighur patients. In Han patients, EGFR-sensitive mutations occurred in female, never-smoking patients with well-differentiated tumors; but for Uighur patients only smoking history showed an obvious correlation.

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