Effect of long non-coding RNA 114227 on gastric cancer cell proliferation and migration / 中南大学学报(医学版)

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.

Molecular cloning and characterization of gene virD[STHX]4 in Helicobacter pylori / 临床检验杂志

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.

Effect of GREM[STHZ]1 on the proliferation and metastasis of gastric cancer cells / 临床检验杂志

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.

Effects of lncRNA LINC00473 on proliferation and migration of human gastric cancer cells / 临床检验杂志

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.

Inhibitory effects of aloin on growth activity and hemolytic toxin expression of Staphylococcus aureus / 临床检验杂志

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.

Isobaric tags for relative and absolute quantitation for preliminarily screening of differential proteins in hippocampus of Ts65Dn mice / 中华围产医学杂志

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.

Expression of ceruloplasmin in Down syndrome babies and their mothers / 中华围产医学杂志

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.

Slienced TROP2 gene inhibits the migration and invasion of gastric cancer BGC-823 cells in vitro / 基础医学与临床

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.

The distribution and structure of class Ⅰ integron in the multidrug-reisistant Pseudomonas aeruginosa / 中华检验医学杂志

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.

Cloning,Expressing and Analysis of Helicobacter pylori NCTC 11637 hp0525 Gene and Study the Influence of Proliferation of SGC-7901 Cell / 中国生物工程杂志

Objective:Helicobacter pylori is one of the human pathogens causing gastric ulcers and cancers.A key virulence factor of H.pylori is the Cag pathogenicity island,which encodes a type IV secretion system.HP0525 is an essential component of the Cag system and acts as an inner membrane associated ATPase.To construct recombinant plasmid containing hp0525 gene of Helicobacter pylori(H.pylori)NCTC 11637,and analysis of sequence nucleic acid,express it in E.coli BL21 and study the influence of the HP0525 protein on proliferation of SGC-7901 cells.Methods:H.pylor hp0525 gene was amplified from the genome DNA by PCR,then operated T-A cloning and sequenced.The hp0525gene fragment was inserted directionally into vector pMD18-T to construct recombinant clones of hp0525 and was sequenced.The recombinant plasmid was transformed into E.coli BL21for expressing under induction of IPTG.Purify the expressed protein by Ni2+-NTA column chromatography.Expressed product was analyzed by Western blot and MALDI-TOF.Added the purified protein into SGC-7901 cells,the effect of cell proliferation in SGC-7901 cells induced by the recombinant protein was observed by MTT.Results:A 993 base pairs long hp0525 gene,which encodes a product of 330 amino acid,was obtained using PCR method and was cloned into pMD18-T vector successfully.The sequence analysis for hp0525 showed that it shares 97%~99% homology with other strains in Gene bank.SDS-PAGE showed a protein band with a relative molecular weight of 36 000,which was consistent with the expectation.The expressed product reached a purity of 97% after Ni2+-NTA column chromatography.The protein after dialyzed and annealed,was co-cultured with SGC-7901 cells,The protien of different concentration co-cultured with SGC-7901 cells for different times,found that the protein in low concentration stimulates proliferation of cells,to achieve some concentration,it inhibits proliferation of cells along with multiplication of the concentration of the protein;The protein inhibits proliferation of cells relay on the extension of time and concentration.Conclusion:It is indicated that the correct hp0525 gene was obtained and expressed in E.coli BL21.High purified protein was obtained by Ni2+-NTA column chromatography,the protein can inhibits proliferation of SGC-7901cells,and T-ATPase activity which posed a basis for further researching on its biological function.

THE IMMUNOLOGIC PROTECTION OF SELENIUM ON GASTRIC MUCOSAL LESIONS OF MICE INFECTED BY HELICOBACTER PYLORI / 营养学报

Objective: To study the immunologic protection of selenium on gastric mucosal lesions of BALB/C mice infected by Helicobacter pylori (HP). Method:The model was built by ig HP in BALB/C mice and then effects of selenium on gastric mucosal lesions and the CD4、CD8 cells were observed.HP was examined by both of bacterial culture and rapid usease test (RUT). Results:1.0 ?g/g bw selenium could effectively prevent gastric mucosal damages induced by HP. HP could decreased the ratio of CD4 to CD8,but selenium could enhance the activity of CD4 cell and increase the ratio. Conclusion: Se can effectively prevent the gastric mucosal damages of mice infected by HP. One of the mechanisms is that Se can enhance the CD4 activity and increase CD4 /CD8.

Expression and relationship among CD44V6, p16 and PCNA in gastric mucosal lesions with Helicobacter pylori infection / 中华检验医学杂志

Objective To study the expression and relationship among CD44V6,p16 and PCNA in gastric mucosal lesions with HP infection. Methods Expression of CD44V6,p16 and PCNA were investigated in 114 gastric mucosal lesions by use of immunohistochemistry.HP was examined by both Warthin -Starry method and RUT. Ruselts Comparing HP positive group (P

Genotyping of SCCmec and Detection of Antibiotic-resistant Genes in Meticillin-resistant Staphylococcus aureus / 中华医院感染学杂志

OBJECTIVE To investigate the staphylococcal cassette chromosome mec(SCCmec) genotype characteristics and antibiotic-resistant genes in meticillin-resistant Staphylococcus aureus(MRSA) isolated from Lianyungang.METHODS The SCCmec of clinically isolated MRSA strains were genotyped with a novel multiplex PCR strategy reported by Zhangetal.Antibiotic-resistant genes of aac(6′)/aph(2″),aph(3′)Ⅲ,tetM,erm,TEM,and ant(4′,4″) were analyzed by traditional PCR.RESULTS The isolates were almost SCCmec Ⅲ positve,only one isolate couldn′t be typed.The positive rates of aac(6′)/aph(2″),aph(3′)Ⅲ,tetM,and erm were 98%,46%,72% and 86%,respectively.TEM and ant(4′,4″) tested were all negative.CONCLUSIONS Almost all genotypes of MRSA prevailing in Lianyungang carry the SCCmec Ⅲ gene.There are high positive percentages of antibiotic-resistant genes of aac(6′)/aph(2″),aph(3′)Ⅲ,tetM and erm in the isolates.The novel multiplex PCR strategy recommended by Zhang et al can be applied into genotyping study of MRSA SCCmec effectively.

Gene cloning and characterization of hopX outer membrane protein from Helicobacter pylori / 中国免疫学杂志

Objective:To clone the outer membrane protein hopX gene of Helicobacter pylori(Hp)and to perform sequencing and analysis of biological information.Methods:Polymerase chain reaction(PCR)was used to amplify the hopX gene from Hp chromosomal DNA.Then the target gene was digested by restricted endonuclease enzyme of BamH I,and inserted into the prokaryotic expression vector pQE30 digested by corresponding restricted endonuclease enzyme.The recombinant vector was used to select and transform for nucleotide sequence analysis.The biological property at the amino acid level was analyzed by Omiga 2.0 and Antheprot v 5.0.The transformant colony was induced with IPTG and the fusion protein was analyzed by SDS-PAGE and Western blot.Results:The recombinant plasmid was constructed.DNA sequence analysis showed the sequence of hopX was 1 284 bp.The homology of the strains in nucleotide acid was 96%～97%.Their homogeneity in the amino acids was 97%～99%.We get a GeneBank accession number EF208122.Omiga 2.0 software predicted its relative molecular mass(Mr.)was 47 kD and possessed good antigencity.The expressed product contained about 37% of total somatic proteins and Western blot method showed good antigenicity of the recombinant protein.Conclusion:A confirmed gene hopX has been obtained,providing a good foundation for recombination,expression and related study.The corresponding peptide of the gene performed the structural characteristics of some typical antigen molecules,which suggest that it might be a novel vaccine candidate.

Detecting of gene oipA of normal and clinical Helicobacter pylor strains and comparing of their nucleotide sequences / 中国免疫学杂志

Objective:To detect the oipA gene of Helicobacter pylori(Hp)strains NCTC11637 and Hp1,Hp2 isolated from clinical biopsies,analyze their nucleotide sequences and make a homologous comparison of nucleotide with Hp 26695.Methods:The oipA gene was detected with PCR in Helicobacter pylori(Hp)strains NCTC11637 and Hp1,Hp2 isolated from clinical gastric biopsies after routine culture.Then PCR products were sent out for nucleotide sequence analysis and compared with Hp 26695.Results:The sequence of the aim gene was obtained in NCTC11637 and Hp1,Hp2 and was made a homologous comparison of nucleotide with 26695.The number of mutation of NCTC11637,Hp1 and Hp2 and was 48,48,50 respectively.The identity was 94%,94% and 94% respectively,while the strain Hp1 was most identical to 11637 as much as 100%.The homology of Hp2 and 11637 was 97%.Conclusion:Hp1,Hp2 and NCTC11637 expresse gene oipA,but the sequences of gene oipA of different strains are distinct.