Objective To study metabonomics in the urine of rats of different genders by magnetic resonance (MR) with 2 normalization methods. Methods Different normalization methods such as mean-centering not scaling (Ctr) and unit variance scaling (UV) were used before orthogonal to partial least squares discriminant analysis(OPLS-DA). Distinguished metabolites in the urine of different gender rats were analyzed by calculating the correlation coefficients. Results The data normalized by Ctr before OPLS-DA analysis revealed high degree conception metabolites in the urine such as valine, alanine, acetate, ornithine, aminohippurate, phenylethylamine, cytosine, citrate, dimethylamine, allantoin, methylamine, fumarate and one unknown metabolite whose chemical shift was δ4.14. Data normalized by UV before OPLS-DA analysis revealed the above 12 high degree conception metabolites except citrate, and also low degree conception metabolites such as thiamine, creatinine, formate and one unknown metabolite whose chemical shift was δ2.92.Conclusion Unit variance scaling is a more effective normalization method in metabonomic analysis.

ObjectiveTo evaluate the relationship and significant between low molecular-weight protein (LMP) methylation and expression,and Uyghur women cervical lesions with human papillomavirus (HPV) infection.MethodsGenetic information was obtained from the GenBank database,specialized software was used to scan gene promoter regions,and CpG island fragment specific primers was designed,gene methylation and CpG site sequences related information were gained by the PCR amplification,vector cloning and sequencing of the bisulfate-modified cervical cancer cell DNA.Methylation status of LMP was quantitative evaluated by Sequenom MassARRAY DNA in 78 subjects with different cervical lesions; mRNA and protein of LMP2 and LMP7 were evaluated by RT-PCR and immunohistochemistry.HPV infection status determined use HPV gene chips.ResultsGene promoter region CpG fragments methylation sequencing it was detected that selected CpG sites of cervical cancer cell genome LMP7 had methylation.However no methylation site was found in gene promoter region of LMP2.The percent of LMP7 methyiation was increased steadily with the severity of cervical lesions.showing 0.0652±0.0488,0.0728+0.0548 and 0.1864+0.0893 of which with normal control,CIN and CSCC (P＜0.01).LMP7 was significantly reduced in CSCC and CIN compared with normal cervical epithelium,and its mRNA expression consistent with the results of proteins,while no significant difference in LMP2 expression.Moreover,the methylated proportion of LMP7 was negatively associated with the protein expression in cervical lesions ( F =8.69,P =0.035 ).Stratified analysis indicated that the percents of LMP7 methylation in subjects with HPV16 positive still increased( t=1.996,P=0.049).ConclusionOur findings indicated that LMP7 methylation was significantly associated with cervical lesions and HPV infection.

ATP-Binding Cassette Sub-Family B Member 2 ; ATP-Binding Cassette Transporters ; metabolism ; ATP-Binding Cassette, Sub-Family B, Member 3 ; Calnexin ; metabolism ; Calreticulin ; metabolism ; Carcinoma, Squamous Cell ; metabolism ; pathology ; virology ; Cervical Intraepithelial Neoplasia ; metabolism ; pathology ; virology ; Cysteine Endopeptidases ; metabolism ; Female ; HLA-A Antigens ; metabolism ; Human papillomavirus 16 ; isolation & purification ; Humans ; Lymphatic Metastasis ; Papillomavirus Infections ; Proteasome Endopeptidase Complex ; metabolism ; Uterine Cervical Neoplasms ; metabolism ; pathology ; virology

OBJECTIVETo analyze the metabonomic (1)H-MRS of plasma samples from patients with esophageal cancer and healthy controls applying different pattern recognition methods, and to explore the potential of application of (1)H-MR-based metabonomics in clinical research.

METHODS(1)H-MR was performed on plasma samples from 109 EC patients and 50 health controls to analyze the metabonomic variation between EC patients and healthy subjects and the corresponding (1)H-MRS were recorded on Varian Unity ANOVA 600 MHz to perform principal components analysis (PCA), partial least squares discriminant analysis (PLS-DA), orthogonal partial least squares discriminant analysis (OPLS-DA), respectively.

RESULTSOPLS-DA analysis could correctly separate almost all the plasma samples from EC patients and health controls, better than both the PCA and PLS-DA. The plasma levels of leucine, alanine, isoleucine, valine, glycoprotein, lactate, acetone, acetate, choline, isobutyrate, unsaturated lipid, VLDL, LDL, 1-methylhistidine were significantly decreased in EC patients (r total > 0.27, P < 0.05), while that of dimethylamine, α-glucose, β-glucose, citric acid increased in the EC patients (r total < -0.27, P < 0.05).

CONCLUSIONSThe analysis of metabonomic (1)H-MRS of plasma samples by OPLS-DA method can eliminate the influence of non-experimental factors and decrease the heterogeneity of samples. It is useful and of great potential for application in clinical diagnosis and research of esophageal cancer.

Adult ; Aged ; Blood Chemical Analysis ; China ; ethnology ; Discriminant Analysis ; Esophageal Neoplasms ; blood ; Female ; Humans ; Least-Squares Analysis ; Magnetic Resonance Spectroscopy ; Male ; Metabolomics ; Middle Aged ; Plasma ; metabolism ; Principal Component Analysis

OBJECTIVE: To investigate gender variability in the metabolic serum and urinary profile of healthy Han population in Xinjiang. METHODS: Serum and urinary samples from 92 healthy Han people in Xinjiang were tested by magnetic resonance based metabonomics and pattern recognition analysis performed with orthogonal partial least-squares discriminant analysis (OPLS-DA). The quality of the model was described by parameter R(2)X, R(2)Y, and Q(2). RESULTS: The serum in males had higher levels of very low density lipoprotein, low density lipoprotein, unsaturated lipids, creatinine and acetone than in females, whereas females had higher levels of citrate, choline, glucose and amino acids (including isoleucine, leucine, valine, alanine, citrulline, lysine, methionine, glutamate, phenylalanine, threonine, tyrosine, 1-methyl histidine and glycine) than in males. The urine of males had higher levels of formate, malonic acid, taurine, creatinine than that of females, while females had higher levels of hippurate, γ-aminobutyric acid, succinate, citrate and glutamate than males. The model parameters of serum were R(2)X=0.64, R(2)Y=0.70, and Q(2)=0.67, and those of urine were R(2)X=0.17, R(2)Y=0.70, and Q(2)=0.44. CONCLUSION The blood and urine from Han population in Xinjiang contain a variety of gender related metabolites, which plays an important role in the research of clinical metabonomics.
Amino Acids ; blood ; urine ; Blood Chemical Analysis ; China ; ethnology ; Citric Acid ; blood ; urine ; Creatinine ; blood ; urine ; Female ; Humans ; Lipids ; blood ; urine ; Lipoproteins ; blood ; urine ; Magnetic Resonance Spectroscopy ; Male ; Metabolome ; Metabolomics ; methods ; Sex Factors ; Urinalysis

BACKGROUNDIt has been confirmed that defective expression of human leukocyte antigen class I (HLA-I) molecules can contribute to the immune evasion of cancer cells in some types of cancer. The aim of this study was to examine the expression of HLA class I antigen and the antigen-processing machinery (APM) components in esophageal squamous cell carcinoma (ESCC) and their role in high risk human papillomavirus (HPV) infection, and to analyze their association with histopathological characteristics in the Kazak ethnic group.

METHODSA total of 50 formalin-fixed, paraffin-embedded ESCC lesions were collected from the First Affiliated Hospital of Xinjiang Medical University, China. The expression levels of HLA-I antigen and APM components were determined by immunohistochemistry; the HPV DNA were detected using polymerase chain reaction (PCR).

RESULTSA high frequency of down-regulation or loss of expression of HLA and APM components were found in esophageal cancer in Kazak people. HLA-I, TAP1, CNX, LMP7, Erp57, Tapasin and ERAP1 were down-regulated in 68%, 44%, 48%, 40%, 52%, 32% and 20% of ESCC lesions then, respectively. The loss of expression of HLA-I antigen was significantly correlated with part of the APM components and positively correlated with high risk HPV16 infection. TAP1, CNX, LMP7, Erp57 and Tapasin loss were significantly associated with tumor grading, lymph node metastasis and depth of invasion (P < 0.05).

CONCLUSIONOur results suggest that APM component defects are a mechanism underlying HLA-I antigen down-regulation in ESCC lesions, and indicate that the loss expression of HLA-I and APM components will become an important marker of ESCC and analysis of HLA-I and APM component expression can provide useful prognostic information for patients with ESCC from the Kazak ethnic group.

ATP-Binding Cassette Sub-Family B Member 2 ; ATP-Binding Cassette Transporters ; genetics ; metabolism ; Adult ; Aged ; Aminopeptidases ; genetics ; metabolism ; Antigen Presentation ; genetics ; physiology ; Calnexin ; genetics ; metabolism ; Esophageal Neoplasms ; metabolism ; Female ; Histocompatibility Antigens Class I ; genetics ; metabolism ; Human papillomavirus 16 ; genetics ; Humans ; Immunohistochemistry ; In Vitro Techniques ; Male ; Membrane Transport Proteins ; genetics ; metabolism ; Middle Aged ; Minor Histocompatibility Antigens ; Polymerase Chain Reaction ; Proteasome Endopeptidase Complex ; genetics ; metabolism ; Protein Disulfide-Isomerases ; genetics ; metabolism

OBJECTIVETo investigate the relationship and significance between endoplasmic reticulum protein 57 (ERp57) gene promoter region methylation with the pathogenesis of cervical lesions in Uighur women.

METHODSThe special software was used to design specific primers of CpG island fragments of ERp57 gene promoter and bisulfite-modified SiHa cancer cell DNA for PCR amplification, cloning and sequencing the target fragments to obtain relevant information of CpG methylation in the gene base sequencs. Seventy-eight fresh tissues of CIN, CSCC and normal control were collected, and the methylation level of ERp57 gene promoter regions in different cervical lesions were identified using Sequenom MassARRAY(DNA) technology.

RESULTSERp57 gene corresponding target fragment contained the 18 CpG sites. All of the CpG sites methylation occurred in SiHa cervical cancer cell genomic DNA. The analysis of the data resulted from the quantitative analysis of single CpG site methylation by Sequenom MassARRAY platform showed that the methylation level between three CpG sites (CpG_1, CpG_5 and CpG_7) from CpG_1, CpG_2, CpG_3.4, CpG_5, CpG_6, CpG_7, CpG_8 and CpG_ 9 had significant differences in the CSCC, CIN or control groups.

CONCLUSIONSAlthough the global methylation level of the ERp57 gene promoter is higher in CSCC than that in CIN and normal control tissues in Uighur women, hypermethylation occurs only in certain CpG islands and sites. This indicates that the regulation of expression by DNA methylation is not CpG island-specific, but varies for individual CpG sites, and may explain to a certain extent the epigenetic mechanisms regulated by Erp57 gene expression.

Adult ; Aged ; Asian Continental Ancestry Group ; ethnology ; genetics ; Carcinoma, Squamous Cell ; genetics ; Cell Line, Tumor ; Cervical Intraepithelial Neoplasia ; genetics ; CpG Islands ; genetics ; DNA Methylation ; Female ; Humans ; Middle Aged ; Promoter Regions, Genetic ; genetics ; Protein Disulfide-Isomerases ; genetics ; Uterine Cervical Neoplasms ; genetics

OBJECTIVETo study the relationship between TAP (transporter associated with antigen processing) gene promoter regional methylation level and cervical lesions with HPV infection in Uyghur women.

METHODSA specialized software was used to design specific primers of CpG island fragments of TAP1 and TAP2 gene promoter for PCR amplification, bisulfitemodified SiHa cancer cell DNA for PCR amplification, cloning and sequencing analysis to obtain the relevant information on the gene base sequence methylation of CpG sites. Seventy-eight fresh cervical tissue samples from Uyghur women with cervicitis (number = 15), cervical intraepithelial neoplasia (CIN, number = 30) and cervical squamous cell carcinoma (number = 33) were collected. The methylation level of TAP1 and TAP2 gene promoter regions was detected using MassArray DNA technology. HPV infection status was determined by HPV gene chips. The relationship between CpG-island methylation of gene promoter regions and HPV infection was then analyzed.

RESULTSEach TAP1 and TAP2 gene corresponding target fragment contained 23 and 8 CpG sites. There were 5 and 8 CpG sites methylation occurred in SiHa cervical cancer cells genomic DNA respectively. The TAP1 methylation level increased steadily with the severity of cervical lesions. The methylation levels in cervical squamous cell carcinoma and CIN (0.048 ± 0.039 and 0.037 ± 0.026, respectively) were higher than that of normal cervical tissue (0.035 ± 0.029, P < 0.05). Although TAP2 gene methylation level also demonstrated similar changes, the difference however was not statistically significant (P > 0.05). HPV gene chip detected 13 HPV genotypes, with HPV16 infection rate being 66.7% (52/78). The methylated proportion of TAP1 positively correlated with HPV16 infection (χ(2) = 6.08, P = 0.039).

CONCLUSIONTAP1 methylation is a remarkable phenomenon occurring in a range of cervical lesions and significantly associated with cervical HPV infection.

ATP-Binding Cassette Sub-Family B Member 2 ; ATP-Binding Cassette Transporters ; genetics ; ATP-Binding Cassette, Sub-Family B, Member 3 ; Adult ; Aged ; Asian Continental Ancestry Group ; genetics ; Carcinoma, Squamous Cell ; genetics ; virology ; Cervical Intraepithelial Neoplasia ; genetics ; virology ; CpG Islands ; genetics ; DNA Methylation ; Female ; Human papillomavirus 16 ; Humans ; Middle Aged ; Papillomavirus Infections ; Promoter Regions, Genetic ; Uterine Cervical Neoplasms ; genetics ; virology ; Uterine Cervicitis ; genetics ; virology

OBJECTIVETo explore the relationship between human papillomavirus(HPV) infection and expression of human leukocyte antigen class I (HLA-I) family genes (HLA-A, B and C) in cervical cancers of Uighur women, and to investigate their effect on cervical cancer progression.

METHODSFresh tissue samples of 78 Uighur women with cervical squamous carcinoma, cervical intraepithelial neoplasia (CIN) or benign cervicitis were selected. HLA-A, B and C expression and HPV infection were analyzed using RT-PCR and HPV gene chips, respectively.

RESULTSThere was a tendency of increasing the total loss of HLA-A, B and C mRNA as the cervical lesions became more aggressive. Loss of HLA-I mRNA in CIN (I, II and III) and cervical squamous carcinoma was 70.0% (14/20) and 84.8% (39/46) respectively. Poorly differentiated cervical carcinomas had the highest HLA-I expression loss (90.6%). In contrast, HLA-I mRNA loss was seen in only 8% of cases of cervicitis. Moreover, it was found that high risk HPV 16 infection was strongly correlated with the loss HLA-I mRNA expression (r = 0.803, P < 0.01).

CONCLUSIONSThe loss of HLA-I gene expression is strongly correlated with HPV-16 infection, and may serve as a biomarker of cervical cancer progression in Uighur women.

Adult ; Aged ; Carcinoma, Squamous Cell ; ethnology ; genetics ; immunology ; virology ; Cervical Intraepithelial Neoplasia ; ethnology ; genetics ; immunology ; virology ; China ; ethnology ; Female ; HLA Antigens ; genetics ; metabolism ; HLA-A Antigens ; genetics ; metabolism ; HLA-B Antigens ; genetics ; metabolism ; HLA-C Antigens ; genetics ; metabolism ; Human papillomavirus 16 ; isolation & purification ; Humans ; Middle Aged ; Papillomavirus Infections ; ethnology ; genetics ; immunology ; virology ; RNA, Messenger ; metabolism ; Uterine Cervical Neoplasms ; ethnology ; genetics ; immunology ; virology ; Uterine Cervicitis ; ethnology ; genetics ; immunology ; virology

Objective To evaluate and discuss the changes of biomarkers of abnormal Savda syndrome rat model in Uyghur traditional medicine by external feature observation and urine metabolomics assessment. Methods The abnormal Savda syndrome rat model was established according to the theory of Uyghur traditional medicine. Its external characteristics such as hair, tongue, sleep, feces, emotion and weight growth rate were observed and scored, and their urine was detected and analyzed by proton nuclear magnetic resonance(1H-NMR)spectroscopy. Results Compared with the healthy control group,there were significant changes in the external features in the abnormal Savda syndrome rat model group,including dry and hard stools,reduced urine output and darker color,dry fur,dark purple tongue with ecchymosis, and decreased weight growth rate. Moreover,23 urinary metabolites were significantly reduced,including propionate,lactic acid, pyruvic acid, acetic acid, alanine, acetamide, glycoprotein, acetone, methyl guanidine, sarcosine, ornithine, glycine, creatine, creatinine, aminoanhydride, β-galactose, urocanate, tyrosine, phenylalanine, hippuric acid, aminohippuric acid,formic acid and lysine. However,urea,citric acid,allantoin and α-ketoglutaric acid were significantly increased. Conclusions During the development process of Savda syndrome, there are not only abnormal changes in external appearance in the model rats, but also evident changes of many internal metabolic pathways. The obvious abnormalities of the urine metabolites may be related to the biological mechanisms of abnormal Savda syndrome.