m6 A-Dependent Modulation via IGF2BP3/MCM5/Notch Axis Promotes Partial EMT and LUAD Metastasis.

The importance of mRNA N6-methyladenosine (m6 A) modification during tumor metastasis is controversial as it plays distinct roles in different biological contexts. Moreover, how cancer cell plasticity is shaped by m6 A modification is interesting but remains uncharacterized. Here, this work shows that m6 A reader insulin like growth factor 2 mRNA binding protein 3 (IGF2BP3) is remarkably upregulated in metastatic lung adenocarcinoma (LUAD) and indicates worse prognosis of patients. Interestingly, IGF2BP3 induces partial epithelial-mesenchymal-transition (EMT) and confers LUAD cells plasticity to metastasize through m6 A-dependent overactivation of Notch signaling. Mechanistically, IGF2BP3 recognized m6 A-modified minichromosome maintenance complex component (MCM5) mRNAs to prolong stability of them, subsequently upregulating MCM5 protein, which competitively inhibits SIRT1-mediated deacetylation of Notch1 intracellular domain (NICD1), stabilizes NICD1 protein and contributes to m6 A-dependent IGF2BP3-mediated cellular plasticity. Notably, a tight correlation of the IGF2BP3/MCM5/Notch axis is evidenced in clinical LUAD specimens. Therefore, this study elucidates a critical role of m6 A modification on LUAD cell plasticity in fostering tumor metastasis via the above axis, providing potential targets for metastatic LUAD.

Cytoplasmic Clusterin Suppresses Lung Cancer Metastasis by Inhibiting the ROCK1-ERK Axis.

Clusterin (CLU) is a heterodimeric glycoprotein that has been detected in diverse human tissues and implicated in many cellular processes. Accumulating evidence indicates that the expression of secreted CLU correlates with the progression of cancers. However, the molecular mechanisms underlying its tumor-suppressive roles are incompletely uncovered. In this study, we demonstrate that precursor CLU is widely downregulated in lung cancer tissue, in which secretory CLU proteins are slightly decreased. Impressively, overexpressing CLU potently inhibits the migration, invasion and metastasis of lung cancer cells, whereas silencing CLU promotes this behavior; however, it appears that secretory CLU fails to exert similar anti-metastatic effects. Interestingly, the cytoplasmic precursor CLU binds ROCK1 to abrogate the interaction between ROCK1 and ERK and impair ERK activity, leading to the suppression of lung cancer invasiveness. Meanwhile, the expression of CLU was remarkably diminished in lung cancer bone metastasis loci when compared with subcutaneous tumors in the mouse model and hardly detected in the bone metastasis loci of lung cancer patients when compared with the primary. These findings reveal a novel insight into the function and regulation of cytoplasmic CLU in lung cancer, which might be a potential target for the diagnosis and treatment of metastatic lung cancer.

Effect of molecular weight on the properties of chitosan films prepared using electrostatic spraying technique.

Chitosan (CH) films were prepared with different number-average molecular weight (MW, ca. 6.55, 12.93 and 47.70 kDa) and concentration (0.5, 0.75, and 1.0%) using electrostatic spraying (ES) technique. The effects of MW on film-forming solution properties, ES-atomization performances, and film characteristics were investigated. With increase of MW, conductivity, viscosity, surface tension, and contact angle of CH film-forming solution was raised due to the increases of proportions of amine-groups and degrees of CH chain entanglements. Spray cone angle was generally higher at 12.93 kDa, while the average median diameters of film-forming solution droplets were in the range of 6.3-12.0 µm, indicating well atomization effect. Water barrier property and tensile strength of CH films were improved along with increased MW, which was in accordance with the more completed, rougher and more crystalline microstructures implying by SEM, AFM and XRD. However, film antibacterial capacities against E.coli and L. innocua were descended with increased MW. Considering the overall properties, CH film prepared by 47.70 kDa at 0.5% possessed the best performances.

Characterization of the Multi-Drug Resistance Gene cfr in Methicillin-Resistant Staphylococcus aureus (MRSA) Strains Isolated From Animals and Humans in China.

We investigated cfr-positive and -negative MRSA strains isolated from animals and humans in different geographical areas of China, from 2011 to 2016. Twenty cfr-positive strains (15.6%) were identified from 128 MRSA strains including 17 from food animals and three from humans. The resistance rates and prevalence of the tested antibiotic resistance genes (ARGs) in the cfr-positive MRSA isolates were higher than that in the cfr-negative MRSA isolates. All cfr-positive MRSA isolates were co-carrying fexA and ermC, and had significantly higher optrA incidence rate vs. the cfr-negative isolates (P < 0.05). In addition, multilocus sequence typing (MLST) assays showed that ST9 and spa-type t899 were the most prevalent ST and spa types in the study strains. However, all of the 20 cfr-positive and 10 randomly selected cfr-negative MRSA isolates were clonally unrelated as determined by pulsed-field gel electrophoresis (PFGE) analyses. Importantly, the cfr gene was successfully transferred to a recipient Staphylococcus aureus strain RN4220 from 13 of the 20 cfr-positive MRSA isolates by electroporation. Among these 13 cfr-positive MRSA isolates, two different genetic contexts surrounding cfr were determined and each was associated with one type of cfr-carrying plasmids. Of note, the predominant genetic context of cfr was found to be a Tn558 variant and locate on large plasmids (∼50 kb) co-harboring fexA in 11 of the 13 MRSA isolates. Furthermore, the cfr gene was also identified on small plasmids (∼ 7.1 kb) that co-carried ermC in two of the 13 MRSA isolates. Our results demonstrated a high occurrence of multi-drug resistance in cfr-positive MRSA isolates, and the spread of cfr might be attributed to horizontal dissemination of similar cfr-carrying transposons and plasmids.

Fecal Clostridium symbiosum for Noninvasive Detection of Early and Advanced Colorectal Cancer: Test and Validation Studies.

OBJECTIVE: Current non-invasive early detection of colorectal cancer (CRC) requires improvement. We aimed to identified a fecal Clostridium symbiosum-based biomarker for early and advanced colorectal cancer detection. DESIGN: In the test stage, the relative abundance of Clostridium symbiosum (C. symbiosum) was measured by qPCR in 781 cases including 242 controls, 212 colorectal adenoma (CRA) patients, 109 early CRC (tumor restricted to the submucosa) patients, 218 advanced CRC patients. The prediction accuracy was compared to Fusobacterium nucleatum (F. nucleatum), fecal immunochemical test (FIT) and CEA (carcinoembryonic antigen) and validated in an independent cohort of 256 subjects. Current status of the trial:ongoing/still enrolling. Primary endpoint:June, 2017 (Clinicaltrials.gov Identifier NCT02845973). RESULTS: Significant stepwise increase of C. symbiosum abundance was found in CRA, early CRC and advanced CRC (P<0.01). C. symbiosum outperformed all the other markers in early CRC prediction performance. The combination of C. symbiosum and FIT achieved better performance (0.803 for test cohort and 0.707 for validation cohort). For overall discrimination of CRCs, the combination of all above markers achieved the performance of 0.876. CONCLUSIONS: Fecal C. symbiosum is a promising biomarker for early and noninvasive detection of colorectal cancer, being more effective than F. nucleatum, FIT and CEA. Combining C. symbiosum and FIT or CEA may improve the diagnosis power.

Resistance of Helicobacter pylori to antibiotics from 2000 to 2009 in Shanghai.

AIM: To investigate the resistance of Helicobacter pylori (H. pylori) to 6 commonly used antibiotics from 2000 to 2009 in Shanghai. METHODS: A total of 293 H. pylori strains were collected from 2000 to 2009 in Shanghai and tested for their susceptibility to metronidazole, clarithromycin, amoxicillin, furazolidone, levofloxacin and tetracycline using agar dilution. RESULTS: The resistant rates of H. pylori to clarithromycin (8.6%, 9.0% and 20.7%) and levofloxacin (10.3%, 24.0% and 32.5%) increased from 2000 to 2009 in Shanghai. The resistant rate of H. pylori to metronidazole remained stable (40%-50%). Only one strain of H. pylori isolated in 2005 was resistant to tetracycline. All strains were sensitive to amoxicillin and furazolidone. The resistant rate of H. pylori to antibiotics was not related with the sex, age and clinical outcome of patients. CONCLUSION: Resistance of H. pylori to antibiotics plays an important role in making treatment strategies against H. pylori-associated diseases.

High efficacy of 14-day triple therapy-based, bismuth-containing quadruple therapy for initial Helicobacter pylori eradication.

BACKGROUND: The success rate of currently recommended 7-day triple therapy with a PPI plus amoxicillin and clarithromycin has fallen into the unacceptable range. It is urgent to look for a new strategy to treat the infection of Helicobacter pylori. AIMS: To observe the efficacy of triple therapy-based, bismuth-containing quadruple therapy for H. pylori treatment. METHODS: A total of 160 patients with functional dyspepsia who were Hp+ were randomly assigned into two groups. Regimen: Omeprazole 20 mg, Amoxicillin 1.0 g, Clarithromycin 500 mg and Bismuth Potassium Citrate 220 mg, twice a day. Eighty patients received 7-day quadruple therapy and 80 patients received the same therapy for 14 days. Six weeks after treatment, H. pylori eradication was assessed by (13)C-urea breath test. Minimal inhibitory concentrations of metronidazole, clarithromycin and amoxicillin of clinical isolates were determined by the twofold agar dilution method. RESULTS: Fourteen-day therapy led to a significant increase of H. pylori eradication success when compared to 7-day therapy in the intention-to-treat analysis (93.7 vs 80.0%; p = .01), and the per-protocol analysis (97.4 vs 82.0%; p = .0016). The H. pylori resistance rates to metronidazole, clarithromycin and amoxicillin were 42.1, 18.0 and 0%. Fourteen-day therapy was significantly more effective in patients with clarithromycin-resistant strains. Incidences of adverse events were comparable. CONCLUSIONS: Addition bismuth and prolonging treatment duration can overcome H. pylori resistance to clarithromycin and decrease the bacterial load. Fourteen-day triple therapy-based, bismuth-containing quadruple therapy achieved ITT success rate 93% and could be recommended as the first line eradication regimen.

Reduced microRNA-218 expression is associated with high nuclear factor kappa B activation in gastric cancer.

BACKGROUND: Poor expression of microRNAs (miRs) reportedly plays an important role in gastric carcinogenesis. Large-scale microarray assays have indicated that there is significant down-regulation of miR-218 in gastric cancer. miR-218 also was decreased specifically in human papillomavirus-positive cell lines, cervical lesions, and cervical cancer tissues and in bronchial airway epithelium in smokers. However, its role in carcinogenesis remains unclear, especially in Helicobacter pylori (H. pylori)-associated gastric cancer. METHODS: miR-218 levels were evaluated in 20 noncardia gastric cancer tissues, in 10 H. pylori-infected and 8 uninfected normal gastric biopsies, and in the human gastric epithelial cancer cell line AGS using TaqMan quantitative real-time polymerase chain reaction analysis. Pre-miR-218 and anti-miR-218 inhibitors were used to examine the effects of miR-218 expression on cell proliferation and apoptosis. A luciferase reporter assay was used to examine the potential target genes and related pathways. RESULTS: miR-218 expression was reduced significantly in gastric cancer tissues, in H. pylori-infected gastric mucosa, and in H. pylori-infected AGS cells. Overexpression of miR-218 inhibited cell proliferation and increased apoptosis in vitro. Epidermal growth factor receptor-coamplified and overexpressed protein (ECOP), which regulates nuclear factor kappa B (NF-kappaB) transcriptional activity and is associated with apoptotic response, was a direct target of miR-218. Overexpression of miR-218 also inhibited NF-kappaB transcriptional activation and transcription of cyclooxygenase -2, a proliferative gene regulated by NF-kappaB. CONCLUSIONS: H. pylori infection resulted in a decrease in miR-218 expression. The down-regulation of miR-218 has the potential to increase carcinogenesis by losing control of its targets, and it may be correlated with the high transcriptional activity of NF-kappaB that results from H. pylori infection.

[The relationship of mTOR signaling pathway and histone acetylation in human gastric cancer cell lines].

OBJECTIVE: To evaluate the relationship between mammalian target of rapamycin (mTOR) signaling pathway and histone acetylation in cell survival, cell cycle, gene expression and protein level on human gastric cancer cells. METHODS: Human gastric cancer cell lines, MKN45 and SGC7901 were treated with trichostatin A, rapamycin and/or LY294002, a PI3K inhibitor. Cell viability was analyzed by methylthiazolyl tetrazolium. Cell cycle distribution was evaluated by flow cytometry. The transcription level of p21(WAF1) gene was detected by using real-time polymerase chain reaction. Proteins were detected by Western blotting. RESULTS: Cell viability remarkably reduced after treatment by more than two drugs (P< 0.01). Through flow cytometry assessment, MKN45 cells were arrested in G2 phase (P< 0.05), while SGC7901 cells were in G2 or G1 phase (P< 0.05) whether treated with single or more than two drugs. The expression of p21(WAF1) mRNA was remarkably increased in the gastric cancer cells treated with conjoined drugs (P< 0.01). Phosphorylation of Akt, p70S6K and 4E-BP1 was significantly reduced in cells treated with conjoined drugs (P< 0.01). And histone acetylation of H4/H3 was also increased in cells treated with conjoined drugs (P< 0.01). CONCLUSION: mTOR singnaling pathway has an important relationship with histone acetylation in gastric cancer cell lines. There is a co-effect of mTOR inhibitor and histone deacetylase inhibitor on gastric cancer cells.

Folate levels in mucosal tissue but not methylenetetrahydrofolate reductase polymorphisms are associated with gastric carcinogenesis.

AIM: To evaluate whether folate levels in mucosal tissue and some common methylenetetrahydrofolate reductase (MTHFR) variants are associated with the risk of gastric cancer through DNA methylation. METHODS: Real-time PCR was used to study the expression of tumor related genes in 76 mucosal tissue samples from 38 patients with gastric cancer. Samples from the gastroscopic biopsy tissues of 34 patients with chronic superficial gastritis (CSG) were used as controls. Folate concentrations in these tissues were detected by the FOL ACS:180 automated chemiluminescence system. MTHFR polymorphisms were analyzed by PCR-RFLP, and the promoter methylation of tumor-related genes was determined by methylation-specific PCR (MSP). RESULTS: Folate concentrations were significantly higher in CSG than in cancerous tissues. Decreased expression and methylation of c-myc accompanied higher folate concentrations. Promoter hypermethylation and loss of p16(INK4A) in samples with MTHFR 677CC were more frequent than in samples with the 677TT or 677CT genotype. And the promoter hypermethylation and loss of p21(WAF1) in samples with MTHFR 677CT were more frequent than when 677CC or 677TT was present. The 677CT genotype showed a non-significant higher risk for gastric cancer as compared with the 677CC genotype. CONCLUSION: Lower folate levels in gastric mucosal tissue may confer a higher risk of gastric carcinogenesis through hypomethylation and overexpression of c-myc.

T cell immune response is correlated with fibrosis and inflammatory activity in hepatitis B cirrhotics.

AIM: To explore the relationship among interferon-gamma (IFN-gamma) activity, fibrogenesis, T cell immune responses and hepatic inflammatory activity. METHODS: Peripheral blood samples from a total of 43 hepatitis B cirrhotic patients (LC) and 19 healthy controls (NC) were collected to measure their serum levels of IFN-gamma, interleukin-2 (IL-2), soluble interleukin-2 receptor (sIL-2R), interleukin-10 (IL-10) and three serological markers of fibrosis including hyaluronic acid (HA), procollagen type III peptide (PIIIP), and type IV collagen were measured using a double antibody sandwich ELISA. Also, serum total bilirubin (TB) and alanine aminotransferase (ALT) were measured by routine measures. RESULTS: The concentrations of serological markers of fibrosis in patients with active cirrhosis (ALC) were significantly higher than those in stationary liver cirrhosis (SLC) or NC groups. The levels of serological markers in HBeAg-positive patients were significantly higher than those in HBeAg-negative patients. In SLC and ALC patients, a negative linear correlation was found between IFN-gamma levels and the serological markers of fibrosis. IFN-gamma and IL-2 levels in the ALC group were significantly higher than those in the SLC and NC groups, but the statistical difference was not significant between the latter two. In contrast, IL-10 levels in the SLC group were significantly higher than that in the NC group, but no significant difference was found between SLC and ALC groups. The sIL-2R level was elevated gradually in all these groups, and the differences were significant. Positive linear correlations were seen between IFN-gamma activity and ALT levels (r = 0.339, P < 0.05), and IL-2 activity and TB levels (r = 0.517, P < 0.05). sIL-2R expression was positively correlated with both ALT and TB levels (r = 0.324, 0.455, P < 0.05), whereas there was no statistically significant correlation between IL-10 expression and serum ALT and TB levels (r = -0.102, -0.093, P > 0.05). Finally, there was a positive correlation between IFN-gamma and IL-2 levels. CONCLUSION: T cell immune responses are correlated with fibrosis and hepatic inflammatory activity and may play an important role in liver cirrhosis.