Objective To investigate the effects of Helicobacter pylori(Hp)on the proliferation,migration,apoptosis and inflammatory response of human umbilical vein endothelial cells(HUVEC)through activation of STAT3/nuclear factor κB(NF-κB)pathway.Methods HUVEC were divided into control group(without Hp infection)and Hp group(multiplicity of infection=25).Cell morphology was observed with inverted microscopy,proliferation was detected by CCK-8 assay and plate cloning assay,and the migration ability was examined by Transwell migration as-say and wound healing assay.Flow cytometry was used to detect the apoptotic rate.Real-time fluo-rescence quantitative PCR was employed to measure the mRNA expression of cytotoxin-associat-ed gene A(CagA),IL-6,IL-8,IL-1β and TNF-α.Western blotting was applied to determine the protein expression of Cyclin D1,proto-oncogene C-Myc,MMP-2,MMP-9,PCNA,Bax,Bcl-2 and STAT3/NF-κB signaling pathway.Results Hp infection resulted in suppressed proliferation and migration abilities,decreased protein levels of Cyclin D1,PCNA,C-Myc,MMP-2,MMP-9 and Bcl-2,elevated protein levels of Bax,p-STAT3/STAT3,p-NF-KB p65/NF-κB p65,raised apoptotic rate,and significantly increased mRNA levels of IL-6,IL-8,IL-1β and TNF-α(2.71±0.05 vs 1.06±0.41,1.42±0.02 vs 0.92±0.11,2.50±0.29 vs 1.00±0.10,5.34±0.57 vs 1.00±0.16;P＜0.01)when compared with the control group.Conclusion Hp infection inhibits proliferation and migra-tion,and induces apoptosis and inflammatory response in HUVEC through activation of the STAT3/NF-κB pathway.

OBJECTIVES: Gastric cancer is a common cancer of the digestive system. Long non-coding RNA (lncRNA) plays an important role in the formation and development of gastric cancer. This study aims to investigate the effect of long non-coding lncRNA 114227 on biologic behaviors in gastric cancer cells. METHODS: The experiment was divided into 4 groups: a negative control (NC) group, a lncRNA 114227 small interference (si-lncRNA 114227) group, an empty vector (Vector) group, and an overexpression vector (OE-lncRNA 114227) group. The expressions of lncRNA 114227 in gastric mucosa and gastric cancer tissues, gastric mucosal epithelial cells and different gastric cancer strains were determined by real-time reverse transcription PCR (real-time RT-PCR).The proliferation were detected by CCK-8 assay in gastric cancer cells. The epithelial-mesenchymal transformation (EMT) was utilized by Transwell assay, scratch healing assay, and Western blotting in gastric cancer cells. The effect of lncRNA 114227 on proliferation of gastric cancer cells was detected by tumor bearing experiment in nude mice in vivo. RESULTS: The expression level of lncRNA 114227 in the gastric cancer tissues was significantly lower than that in the gastric mucosa tissues, and in 4 kinds of gastric cancer strains was all significantly lower than that in gastric mucosal epithelial cells (all P<0.01). In vitro, the proliferation and migration abilities of gastric cells were significantly reduced after overexpressing lncRNA 114227, and cell proliferation and migration were enhanced after silencing lncRNA 114227 (all P<0.05). The results of in vivo subcutaneous tumorigenesis in nude mice showed that the tumorigenic volume of the tumor-bearing mice in the OE-lncRNA 114227 group was significantly smaller than that of the Vector group, and the tumorigenic quality was lower than that of the Vector group (P<0.05), indicating that lncRNA 114227 inhibited tumorigenesis. CONCLUSIONS The expression of lncRNA 114227 is downregulated in gastric cancer gastric cancer tissues and cell lines. LncRNA 114227 may inhibit the proliferation and migration of gastric cancer cells through EMT process.
Animals ; Mice ; RNA, Long Noncoding/metabolism* ; Stomach Neoplasms/pathology* ; Mice, Nude ; Cell Line, Tumor ; Cell Proliferation/genetics* ; Carcinogenesis/genetics* ; Cell Movement/genetics* ; Epithelial-Mesenchymal Transition/genetics* ; Gene Expression Regulation, Neoplastic ; Apoptosis/genetics*

Objective: Abstract: Objective: cancer cells and its effects on the proliferation and migration of gastric cancer cells. Methods: The expression level of LINC00473 in gastric cancer cells was verified by qRT-PCR system. LINC00473 siRNA segment and overexpression vector were separately transfected into gastric cancer cells by the method of lipofection. The proliferation and migration abilities of gastric cancer cells with LINC00473 knockdown or overexpression in vitro were evaluated by cell counting kit-8 (CCK8) assay, colony formation assay and Transwell migration assay. The expression levels of proteins involved in epithelial-mesenchymal transition (EMT) were examined by western blot analysis. Results: The expression levels of LINC00473 were decreased in gastric cancer cells compared with that in human gastric epithelial cell strain GES-1 (P＜0.05). LINC00473 knockdown cells showed significant increased ability for cell growth (F=163.10, P＜0.01) and colony formation (t=3.29, P＜0.05) compared with the knockdown cells in scramble control. The results of Transwell migration assay showed that LINC00473-knockdown enhanced the migratory abilities of gastric cancer cells (t=4.68, P＜0.05). The knockdown of LINC00473 downregulated E-cadherin expression (t=4.08, P＜0.05) and upregulated N-cadherin (t=5.06, P＜0.01), Snail (t=7.69, P＜0.01) and Vimentin (t=3.82, P＜0.05) expression. Compared with the control group, LINC00473 overexpression cells showed significantly decreased cell growth (F=186.00, P＜0.01) and colony formation ability (t=3.22, P＜0.05). The results of Transwell migration assay showed that LINC00473-overexpression reduced the migratory ability of gastric cancer cells (t=5.52, P＜0.05). The overexpression of LINC00473 enhanced E-cadherin expression (t=2.90, P＜0.05) and reduced the expressions of N-cadherin (t=7.44, P＜0.01), Snail (t=2.78, P＜0.05) and Vimentin (t=4.64, P＜0.01). Conclusion The knockdown of LINC00473 may promote gastric cancer cell proliferation and migration in vitro by regulating EMT.

Objective: To detect the expression of GREM1 gene in gastric cancer cells, investigate its effects on the biological characteristics of gastric cancer cells and evaluate its application value in the diagnosis and prognosis of gastric cancer. Methods: The expression difference of GREM1 in gastric cancer tissues and adjacent normal tissues was analyzed by the database, and the correlation of GREM1 expression levels with the prognosis of gastric cancer patients was evaluated. The expression levels of GREM1 protein in gastric cancer cell lines were detected by western blot. After GREM1 gene in AGS cells was silenced, its effects on the proliferation, migration, epithelial-mesenchymal transition (EMT) and Wnt/β-catenin pathway of AGS cells were detected by the colony formation assay, Transwell and Western blot, respectively. Results: Kaplan-Meier analysis showed that the patients with high expression of GREM1 gene had low overall survival (OS) and progression-free survival (PFS). The expression level of GREM1 protein in AGS cells was the highest in all gastric cancer cell lines (1.967 ± 0.056). The analysis of colony formation assay, Transwell and Western blot showed that the silencing of GREM1 gene could decrease the proliferation and migration of gastric cancer cells (t=22.00; t=29.60; P＜0.01), increase the expression of E-cadherin (t=10.65, P＜0.01), and decrease the expressions of ZEB1 and MMP2 (t=10.74; t=13.67; P＜0.01) and the expressions of β-catenin, Cyclin D1, c-myc, p-GSK3β and PCNA in the Wnt/β-catenin pathway (t=12.65; t=16.21; t=8.74; t=7.75; t=8.42; P＜0.01). Conclusion GREM1 may induce EMT by activating the Wnt/β-catenin pathway, [JP2]and promote the metastasis and growth of tumors, which may be used as a new molecular diagnostic and prognostic marker for gastric cancer.

Objective: To investigate the function of virD4 gene in Helicobacter pylori clinical strain SBK. Methods: The virD4 gene segment was obtained through T-A cloning method. The prokaryotic expression vector pET-28a(＋)-virD4 was constructed and transformed into E. coli Rosetta for the expression by induction of IPTG. The recombinant proteins were obtained and purified by KCl dyeing with gel cutting method, and identified via SDS-PAGE analysis. The purified recombinant virD4 protein was used to immunize mice to produce polyclonal antibodies. The titer of the polyclonal antibodies was tested by ELISA and the antigenic specificity was identified by western blot. The purified recombinant virD4 proteins were co-cultured with GES-1 cells followed by detecting the expression of inflammatory cytokines secretion and the ability of cell proliferation. Results: The full length of virD4 gene was 1 728 bp. The sequence shared quite high homology with the virD4 gene of isolate Shi470. The recombinant prokaryotic expression plasmid pET-28a(＋)-virD4 was successfully constructed. The recombinant virD4 proteins were obtained by IPTG induction and purified via KCl dyeing method. SDS-PAGE showed that the relative molecular weight of recombinant virD4 protein was 63 000. The purified proteins were used to immunize mice to obtain the anti-virD4 polyclonal antibodies with the titer 512 000. The reaction between anti-virD4 polyclonal antibodies and recombinant virD4 proteins was highly specific. The recombinant virD4 protein induced inflammatory cytokines secretion and promoted GES-1 cell proliferation. Conclusion The virD4 gene was successfully cloned and highly expressed in prokaryotic expression system, and its antibodies were prepared. The recombinant virD4 protein can induce cytokine secretion and cell proliferation.

Objective To investigate the inhibitory effects of aloin on the growth of Staphylococcus aureus and its virulence factors α-hemolysin in vitro.Methods Broth dilution was used to measure the minimum inhibitory concentration (MIC) of water-soluble aloin on S.aureus.Agar drilling method was used to observe the size of inhibition zone of aloin for S.aureus.Plasma coagulase test was used to detect the changes of S.aureus coagulase and absorbance was measured to detect the changes of hemolytic activity when S.aureus was exposed to aloin.Real time PCR was used to detect the effects of aloin on the expressions of hla and agrA mRNA.Results The soluble aloin inhibited the growth of S.aureus in a dose-dependent manner.The inhibition zone diameter of a standard strain of S.aureus (ATCC 25923) was 21.5 mm with MIC of 12.5 mg/mL and 17 mm for the clinical isolate SA1.5 with MIC of 15 mg/mL.After treated with soluble aloin,the coagulase titers of ATCC 25923 were 16,4 and 2 for 1/2 MIC,1 MIC and 2 MIC respectively compared with titer 32 of the control group without soluble aloin.The expression of α-hemolysin of S.aureus ATCC 25923 was down-regulated by soluble aloin and the hemolytic activity of S.aureus ATCC 25923 with 1/2 MIC,1 MIC and 2 MIC groups were (77.4 ±3.41) ％,(42.2 ± 2.4) ％ and (38.7 ± 2.4) ％ respectively.The expression levels of hla were 0.020 3 (0.019 6,0.028 8),0.011 6(0.010 6,0.013 1) and 0.033 7(0.020 2,0.042 9) respectively in the 1/2 MIC,1 MIC and 2 MIC group respectively,and there were significant differences among the three groups (H =16.807,P ＜ 0.05).The expression levels of agrA was 0.074 6 (0.066 2,0.098 2),0.020 8 (0.012 2,0.032 6) and 0.021 3 (0.010 2,0.029 6) in the 1/2 MIC,1 MIC and 2 MIC group respectively,and there were significant differences among the three groups (H =16.320,P ＜ 0.05).Conclusion Aloin may inhibit the growth of S.aureus and could effectively inhibit the expression of α-hemolysin.

Objective To explore the molecular mechanism of hypophrenia induced by Down syndrome (DS).Methods Ts65Dn mice were used as DS animal model.Three female mice and three male mice of three to twelve weeks old were mated.Among the 17 first-generation mice alive,five mice remained Ts65Dn trisomy and 12 mice were normal.Five Ts65Dn mice and five normal mice were selected randomly as Ts65Dn group and control group,and bred till 16 to 18 weeks old for experiments.Differential proteins in hippocampus of mice were tested by isobaric tags for relative and absolute quantitation (iTRAQ).Expressions of the differential proteins in Ts65Dn group were detected compared with those in control group.Results A total of 2805 proteins were identified in hippocampus of Ts65Dn group and control group,and significant differences were observed in the expressions of 374 proteins.Compared with those in control group,expressions of 195 proteins increased and 179 reduced in Ts65Dn group.Sorted by P value from low to high,the seven proteins with the lowest P value were uncharacterized protein C2orf47 homolog,isoform 2 of filamin A-interacting protein 1-like,zinc finger protein,isoform 1 of pericentriolar material 1 protein,SEC23 interacting protein,BAG family molecular chaperone regulator 3 and serpin H1.Conclusions Differential proteins are observed in hippocampus of Ts65Dn mice,perhaps closely correlating to neurological defects.The new technology of iTRAQ helps to screen and identify differential proteins in hippocampus.

ObjectiveTo investigate the relationship between ceruloplasmin expression and Down syndrome (DS). MethodsDifferential protein expression in serum of six mothers with DS fetuses and six mothers with healthy fetuses was detected by two-dimensional electrophoresis and matrix assisted laser desorption ionization-mass spectrum,the results were confirmed by Western blot.The levels of serum ceruloplasmin in 11 mothers with DS fetuses,10 mothers with healthy fetuses,11 DS newborns and 10 healthy babies were detected by enzyme-linked immunosorbent assay.The difference between the two groups was compared by two-independent samples t test. ResultsTwenty-nine differential proteins were found in the serum of the mothers with DS fetuses; among which ceruloplasmin increased significantly compared with that in mothers with healthy fetuese with density ratio of 5.43 (t=2.7102,P＜0.05).Western blot results showed that the expression of ceruloplasmin in maternal serum with DS fetuses (0.95 ± 0.24) was higher than that of normal mothers (0.37±0.14) (t=2.9521,P＜0.05) ; while the expression of ceruloplasmin in DS babies' serum (0.74±0.03) was lower than that of normal newborns (0.89±0.06)(t=-2.9515,P＜0.05).The expression of ceruloplasmin in serum of mothers with DS fetuses [(346.5± 111.8) ng/ml] was higher than that of normal mothers [(248.6478.3) ng/ml] (t=2.301,P＜0.05) ; while the expression of ceruloplasmin in DS babies' serum [(166.1 ±55.0) ng/ml] was lower than that of normal newborns [(244.0±36.0) ng/ml] (t=-3.873,P＜0.01). ConclusionsAbnormal maternal and neonatal serum ceruloplasmin level might relate to DS.

Objective To investigate the role of TROP2 in migration and invasion of human gastric cancer cells. Methods Small interfering RNA(siRNA) targeting TROP2 gene was constructed by gene cloning and transfection into gastric cancer cell line BGC-823. The expression of mRNA and protein were detected by Real-time quantitative PCR and Western blot assay after RNA interference. The proliferation was determined by MTT assay. Transwell assay was performed to assess the effect of TROP2 targeted RNA interference on the migratory and invasive properties of gastric cancer in vitro. Results Enzyme digestion analysis and DNA sequencing showed that TROP2 targeted RNA interference recombinant plasmids were successfully constructed. The most effective recombinant plasmid was selected. After transfection, knockdown of TROP2 significantly inhibited the proliferation, migration and invasion of BGC-823 cells in vitro(P <0. 05). Conclusion Interfering and down-regulating TROP2 gene can inhibit migration and invasion of gastric cancer cell line BGC-823 in vitro, indicating that TROP2 gene is a potential target for gastric cancer gene therapy.

Objective To investigate the antimicrobial susceptibility of Pseudomonas aeruginosa (P. aeruginosa) isolated from Zhenjiang area to 13 routinely used antibiotics and identify the structure and dissemination of class Ⅰ integron. Methods K-B test was used to determine the resistant rate of 71 strains of P. aeruginosa. DNA template was extracted by boiling method, PCR method was utilized to detect class Ⅰintegron, and subsequently gene cassettes were analyzed by sequencing. Results The resistant rates to 13 routinely used antibiotics were quite different from 18. 3 to 77.5% among 71 strains of P. aeruginosa. The prevalence of class Ⅰ integron was 38%. These integrons include 5 gene cassettes ( aadB, aac (6) - Ⅱ , PSE-Ⅰ , dfrA17 and aadAS), in which dfrA17 and aadA5 gene cassette were frequently found. Comparing with the negative strains of integron, the positive strains of integron has obviously higher resistance to ten the antibiotics including piporacillin, piperacillin-tazobactam, ceftriaxone, cefepime, ceftazidime, gentamicin,amikacin, tobmmycin, levofloxacin, and ciprofloxacin. Conclusions The resistant rates of P. aeruginosa to 13 drugs were different, and the resistant rates of integron positive strains were obviously higher than integron negative strains, which indicates that integron may play an important role in multidrug reisistance of P. aeruginoosa.