OBJECTIVETo study the effect of alternative splicing form -MAD2beta of mitotic arrest deficient protein 2 (MAD2) on the formation of multidrug resistance in human gastric adenocarcinoma cell SGC7901.

METHODSRNA was extracted from a multidrug resistance cell line SGC7901/ADR. The full-length MAD2beta cDNA was obtained by RT-PCR and cloned into the pUCm-T vector, and then recombined into the eukaryotic expression vector pcDNA3.1 in forward direction. Subsequently, pcDNA3.1/MAD2beta vectors were then transfected into SGC7901 cells by lipofectamine. Sensitivity to drug was detected by MTT assay. Cell cycle alteration and intracellular fluorescence intensity were determined by FACS.

RESULTSA fragment of 0.53 Kb was obtained and confirmed by DNA sequencing which was a new alternative splicing form of MAD2 named as MAD2beta. pcDNA3.1/MAD2beta transfected SGC7901 cells (SGC7901/MAD2beta) were more resistant to ADR, VCR and MMC than the control cells (SGC7901/pcDNA3.1), and also ADR fluorescence intensity of SGC7901/MAD2beta cells was lower (P < 0.05) than that of SGC7901/pcDNA3.1 cells.

CONCLUSIONMAD2beta could increase the multidrug resistance of SGC7901 cell line.

Adenocarcinoma ; metabolism ; pathology ; Alternative Splicing ; Antibiotics, Antineoplastic ; pharmacology ; Antineoplastic Agents, Phytogenic ; pharmacology ; Calcium-Binding Proteins ; biosynthesis ; genetics ; Cell Cycle Proteins ; Cell Line, Tumor ; DNA-Binding Proteins ; biosynthesis ; genetics ; Doxorubicin ; pharmacology ; Drug Resistance, Multiple ; Drug Resistance, Neoplasm ; genetics ; Humans ; Mad2 Proteins ; Mitomycin ; pharmacology ; Repressor Proteins ; Smad2 Protein ; Stomach Neoplasms ; metabolism ; pathology ; Trans-Activators ; biosynthesis ; genetics ; Transfection ; Vincristine ; pharmacology

OBJECTIVETo explore the association of T1270533G polymorphism in the glutathione S-transferase M1 (GSTM1) gene with the susceptibility to nasopharyngeal carcinoma (NPC) and clinical phenotype of NPC in Chinese population. METHDOS: The genomic DNAs were obtained from 27 Chinese subjects, and the single nucleotide polymorphism (SNP) in all the exons and relevant intron-exon boundaries of GSTM1 were determined by PCR and direct sequencing. A case-control study was performed to analyze the SNP site T1270533G (the rare allele frequency is 22.2% in Chinese population) in the coding region by means of tetra-primer amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) and sequencing.

RESULTSequence analysis identified 29 SNPs in GSTM1 gene, among which 13 SNPs presented high linkage disequilibrium with each other. No obvious relations were found between the variation in the coding region T1270533G and the clinical phenotype of NPC (RR=0.170, 95% CI =0.95-0.306 for TT homozygotes).

CONCLUSIONThe missense mutation in the coding region T1270533G of GSTM1 gene that causes an amino acid change does not affect the detoxification function of GSTM1, and the T1270533G polymorphism does not have apparent relations to NPC susceptibility in Chinese subjects in Guangdong Province.

Adult ; Aged ; Base Sequence ; Carcinoma, Squamous Cell ; genetics ; China ; Female ; Genetic Predisposition to Disease ; genetics ; Glutathione Transferase ; genetics ; Humans ; Male ; Middle Aged ; Molecular Sequence Data ; Nasopharyngeal Neoplasms ; genetics ; Polymorphism, Single Nucleotide ; Young Adult

OBJECTIVETo identify potential mutations of retinoschisis 1 (RS1) gene responsible for X-linked retinoschisis (XLRS) in two Chinese families.

METHODSThe 6 exons and flanking intronic regions were analyzed with PCR and direct sequencing.

RESULTSTwo RS1 mutations were identified in the two families, which included 1 frameshift mutation (c.573delG, p.Pro192fs) and 1 missense mutation (c.626G>A, p.Arg209His).

CONCLUSIONTwo RS1 mutations have been identified, among which Pro192fs mutation is discovered for the first time in Chinese population. Above results may enrich our understanding of the clinical manifestations of XLRS and facilitated early diagnosis and genetic counseling for the disease.

Adolescent ; Adult ; Eye Proteins ; genetics ; Female ; Genetic Diseases, X-Linked ; diagnosis ; genetics ; Humans ; Male ; Middle Aged ; Mutation ; Prenatal Diagnosis ; Retinoschisis ; diagnosis ; genetics

BACKGROUNDCoronary slow flow phenomenon (CSFP) is an important, angiographic clinical entity but is lacking non-invasive detecting techniques. This study aimed to elucidate the value of transthoracic Doppler echocardiography (TTDE) in the diagnosis and monitoring of coronary slow flow in left anterior descending (LAD) coronary artery.

METHODSWe consecutively enrolled 27 patients with CSFP in LAD detected by coronary arteriography from August 2009 to April 2010. Thirty-eight patients with angiographically normal coronary flow served as control. Corrected thrombolysis in myocardial infarction (TIMI) frame count (CTFC) was used to document coronary flow velocities. All subjects underwent TTDE within 24 hours after coronary angiography. LAD flow was detected and the coronary diastolic peak velocities (DPV) and diastolic mean velocities (DMV) were calculated.

RESULTSSixty of 65 (92.3%) subjects successfully underwent TTDE. Baseline clinical characteristics were similar between the two groups. Coronary DPV and DMV of LAD were significantly lower in the CSFP group than in the control group ((0.228 ± 0.029) m/s vs. (0.302 ± 0.065) m/s, P = 0.000; (0.176 ± 0.028) m/s vs. (0.226 ± 0.052) m/s, P = 0.000, respectively). There was a high inverse correlation between CTFC and coronary DPV and DMV (r = -0.727, P = 0.000; r = -0.671, P = 0.000, respectively). Receiver operating characteristic (ROC) curve showed that the area under the curve (AUC) was less than one half for coronary DPV (AUC = 0.104) and DMV (AUC = 0.204), respectively.

CONCLUSIONSIn patients with CSFP, there is a high inverse correlation between CTFC and coronary diastolic flow velocities in the LAD coronary artery, as measured by TTDE. The value of TTDE in the monitoring and evaluation of coronary flow in patients with CSFP deserves further investigation.

Adult ; Aged ; Blood Flow Velocity ; Coronary Angiography ; Coronary Circulation ; Diastole ; Echocardiography ; methods ; Echocardiography, Doppler ; methods ; Female ; Humans ; Male ; Middle Aged ; No-Reflow Phenomenon ; diagnostic imaging

Objective:To explore the relationship between single nucleotide polymorphism (SNP) rs4977574 in CDKN2B-AS1 gene and female premature coronary artery disease (pCAD) occurrence. Methods: Our research included 2 groups: pCAD group, n=226 consecutive patients≤65 years of age and Control group, n=79 subjects with matched age,without CAD. The genotype of CDKN2B-AS1 SNP rs4977574 was detected by SNaPshot. Blood levels of total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), triglycerides (TG), uric acid (UA), fasting plasma glucose (FPG) and glycosylated hemoglobin (HbA1c) were examined; relationships between rs4977574 polymorphism and the above parameters were assessed. Results: Compared to Control group, pCAD group had increased blood levels of TG, UA, FPG and HbA1c, P<0.05. With adjusted age, body mass index (BMI), relevant disease history and risk factors, elevated HbA1c (HbA1c>6.2%) obviously increased the risk of female pCAD occurrence (OR=3.35, 95%CI 1.41-8.00, P=0.006). The genotype and allele frequency of rs4977574 were different between pCAD group and Control group, P<0.05. Compared to Control group, pCAD group had the higher frequency of G allele(OR=1.24, 95%CI 1.05-1.48, P=0.019); further analysis found that rs4977574 polymorphism was related to high HbA1c. Compared to AA genotype, GG+GA genotype had the increased incidence of high HbA1c(OR=2.08, 95%CI 1.11-3.89, P=0.022). Conclusion: CDKN2B-AS1 SNP rs4977574 was related to female pCAD occurrence and it was also related to high HbA1c.

OBJECTIVETo investigate the effect of gene Af116609 on gastric cancer multi-drug resistance (MDR) by introducing it into gastric cancer multi-drug resistant (MDR) cell line SGC7901/VCR.

METHODSGene Af116609 was cloned from SGC7901/VCR by RT-PCR and its differential expression between gastric cancer MDR cells and its parental cells was displayed by Northern blot. The gene was introduced to gastric cancer cells by transfection of recombinant eukaryotic expression vector by electroporation. MTT assay in vitro was applied to investigate its effect on multi-drug resistance phenotype of gastric cancer cells.

RESULTSThe full length CDS of gene Af116609, as long as 327 bp, was cloned from gastric cancer MDR cell line SGC7901/VCR and its sequence was coincident with the hypothetical gene Af116609 in GenBank. It was overexpressed in MDR cells than its parental cells at mRNA level. In the MTT assay in vitro, the drug sensitive cells transfected with sense eukaryotic expression vector showed upregulated targeted gene, with increased resistance to vincristine, 5-fliorouracil and arabinoside, and decreased resistance to adriamycin, but no influence on resistance to methotrexate. However, the drug resistant cells transfected with anti-sense eukaryotic expression vector, showed down regulated targeted gene, with less resistance to all the five anticancer drugs to different degrees.

CONCLUSIONGene Af116609 is related to MDR phenotype of gastric cancer cells and may become a candidate molecular target to reverse the MDR of gastric cancer.

Antineoplastic Agents, Phytogenic ; pharmacology ; Autoantigens ; genetics ; Cell Line, Tumor ; Drug Resistance, Multiple ; genetics ; Drug Resistance, Neoplasm ; genetics ; Humans ; RNA, Small Cytoplasmic ; genetics ; Ribonucleoproteins ; genetics ; Stomach Neoplasms ; genetics ; pathology ; Vascular Endothelial Growth Factor A ; biosynthesis ; Vincristine ; pharmacology

OBJECTIVETo investigate the differential expression of RPL6/Taxreb107 between drug-resistant gastric cancer cell line SGC7901/ADR and gastric cancer cell line SGC7901 as well as its correlation with multiple-drug resistance (MDR) in gastric cancer cells.

METHODSTotal RNA was extracted from SGC7901 and SGC77901/ADR, with internal control RT-PCR, Northern blot, gene cloning and expression, construction of eukaryotic expression vector, gene transfection by electroporation. The accumulation and retention of ADR in transiently transfected cell was detected by flow cytometry.

RESULTSThe internal control RT-PCR and Northern blot showed high RPL6/Taxreb107 expression in SGC7901/ADR cell line. Sense and antisense eukaryonic expression vectors demonstrated by double enzyme digestion were successfully transfected into gastric cancer cell line SGC7901 and SGC7901/ADR respectively by electroporation. The accumulation and retention of ADR detected 48 hours after transfection showed that RPL6 gene had shown effect on drug resistance in gastric cancer cell.

CONCLUSIONThe high expression of RPL6/Taxreb107 in drug resistant gastric cancer cell shows its correlation with multiple-drug resistance in gastric cancer.

DNA-Binding Proteins ; metabolism ; Drug Resistance, Multiple ; physiology ; Drug Resistance, Neoplasm ; physiology ; Humans ; Statistics as Topic ; Stomach Neoplasms ; pathology ; Tumor Cells, Cultured

Psoraleae Fructus is the dried and mature fruit of the legume Psoralea corylifolia. It is warm in nature， pungent and bitter in flavor， and attributive to the kidney and spleen meridians. Its main effect include warming the kidney and assisting Yang， absorbing Qi and relieving asthma， warming the spleen and relieving diarrhea， etc.， and it also can for external use of eliminating wind and freckle. Clinically， Psoraleae Fructus is mainly used for the treatment of impotence due to kidney deficiency， soreness of waist and knees， vitiligo， etc. The existing studies have shown that Psoraleae Fructus has a variety of pharmacological effect， such as anti-tumor， anti-oxidant， anti-bacterial， anti-inflammatory， promoting bone growth and protecting cardiovascular. But at the same time， many studies at home and abroad have found that taking Psoraleae Fructus and its compounds for a long time or in large doses can cause liver toxicity， phototoxicity， nephrotoxicity， etc. The most common is liver toxicity， most of the clinical reports on the toxicity of psoralen are caused by drug-induced liver injury events， which limits the clinical use of Psoraleae Fructus and can't exert its proper therapeutic effect. Therefore， it is particularly important to fully understand the toxicological mechanism of liver injury caused by Psoraleae Fructus and its attenuation methods. In this paper， by consulting the domestic and foreign related literatures in recent years that reported the hepatotoxicity of Psoraleae Fructus， the four aspects of clinical report on liver injury， hepatotoxic components， toxicological mechanisms and attenuation methods of Psoraleae Fructus were reviewed， including bile acid stasis and oxidative stress. The hepatotoxicity of Psoraleae Fructus was discussed in terms of reaction， mitochondrial damage， liver fat deformation， etc.， and the attenuation methods of Psoraleae Fructus were summarized from the aspects of compatibility attenuation and processing attenuation， aiming to comprehensively and objectively clarify Psoraleae Fructus. The potential toxicological mechanism of lipid-induced hepatotoxicity and research progress in attenuation were expected to provide a theoretical basis for further study of Psoraleae Fructus hepatotoxicity and clinical rational use of drugs.

OBJECTIVEThe effects of lentivirus-mediated suppression of Cyclin Y (CCNY) expression on the proliferation of laryngeal cancer cells were investigated in vitro.

METHODSThe lentivirus vectors containing a small hairpin RNA (shRNA) to target CCNY were constructed.Hep-2 cells were divided into the following two experimental groups:the negative control group (control lentivirus infected cells) and CCNY knockdown group (CCNY shRNA-expressing lentivirus infected cells). After Hep-2 cells were infected, Real-time PCR was used to measure CCNY expression. The influence of CCNY on the proliferation of laryngeal cancer cells were assessed using MTT and colony formation experiments.Each experiment was performed in triplicate and repeated three times.

RESULTSLentiviruses expressing shRNA against CCNY were constructed and Hep-2 cells were infected with above mentioned lentivirus at MOI (Multiplicity of infection) of 120.Real-time PCR analysis showed that the mRNA expression of CCNY in Hep-2 cells in the knockdown group was significantly decreased (P < 0.05); the mRNA level of CCNY was 75.3% lower in the si-CCNY group than in the si-CTRL group. After 5 days of lentiviral infection, the cell viability was significantly lower in cells infected with the CCNY-shRNA lentivirus compared to cells infected with the control lentivirus following a 6-day incubation. The colony number was decreased by 60% in Hep-2 cells infected with the CCNY-shRNA-lentivirus infected cells following a 10-day incubation.

CONCLUSIONSThe results suggested that lentivirus-mediated downregulation of CCNY expression decreased the proliferation and growth potency of laryngeal cancer cells.Lentiviruses delivering shRNA against CCNY may be a promising tool for laryngeal cancer therapy.

Cell Line, Tumor ; Cell Proliferation ; Cyclins ; Humans ; Laryngeal Neoplasms ; metabolism ; Lentivirus ; genetics ; RNA, Small Interfering ; genetics