Project description:Mid-stream urine was collected from bladder cancer patients prior to surgery. Both tumor tissue and normal bladder mucosa that are located at >3cm away from the tumor edge were obtained by cystoscopy. For the normal controls with haematuria, urine samples were collected from patients who had normal cystoscopic finding and absence of malignancy with >6 months follow-up. All urine samples were centrifuged at 2500 r.c.f. for 20 minutes and the urine supernatant was collected. Total RNA of urine supernatant and frozen tissue was extracted using MirVanaTM PARISTM Kit (Ambion) in accordance with the manufacturerâs recommended protocols. AgilentTM Human miRNA Microarray Chip (Release 13.0, Agilent Technologies, Santa Clara, CA, USA) was used to determine the microRNA expression profiles of the samples.
Project description:Antibody microarray based profiling of twelve urine samples.<br>3 healthy female<br>3 heatlhy male<br>3 female with pancreatic cancer<br>3 male with pancreatic cancer<br>
Project description:We performed genome-wide 5hmC Methylated DNA Capture (5hMethylCap-seq) on one pooled RCC tissue sample (n=3) and the corresponding matched normal kidney tissue (NAT) (n=3), and we also performed 5hMethylCap-seq on one pooled urine sample obtained from RCC patients (n=52) along with another pooled urine sample obtained from control patients without malignancy (n=65). Global 5hmC levels were dramatically reduced in RCC tissues compared to matched normal adjacent kidney tissues, and although we detected low levels of 5hmC in urine samples, we also observed reduction of 5hmC in urine samples compared to tissue samples. Through assessing histone marked regions we found that 5hmC levels were enriched in H3K9me3 marked repressive genomic regions of normal adjacent kidney compared to RCC tissue tissues. Given the lower 5hmC signal in other genomic regions in cancer tissues, this upregulated 5hmC levels in H3K9me3 marked regions were also clearly identified comparing urine samples from RCC patients to control patients without RCC. We used Caki1 and Caki2 RCC cells to established stable cells with low H3K9me3 expression by knocking down the SUV39H1 gene. We found that low global H3K9me3 causes major upregulation of 5hmC at H3K9me3 marked regions and minor downregulation of 5hmC at genebody regions without change global 5mC and 5hmC levels.
Project description:Interventions: Preoperation dehydration and postoperation dehydration group:Urine and blood before and after operation room are collected separately;Preoperation dehydration and postoperation normal group:Urine and blood before and after operation room are collected separately;Preoperation normal and postoperation dehydration group:Urine and blood before and after operation room are collected separately;Preoperation normal and postoperation normal group:Urine and blood before and after operation room are collected separately
Primary outcome(s): Urine color;Urine specific gravity;Urine osmotic pressure;Urinary creatinine concentration;Postoperative complications and mortality
Study Design: Case series
Project description:Podocytes form filtration barrier through foot process around glomerualar basement membrane and selectively permit permeability of molecular smaller than albumin. Diabetes can cause podocyte pathological changes leading to high urine albumin level. Diabetic mouse model OVE26 has extremly high urine albumin and previously studies indicated its podocyte damaged. Here we try to find the key genes change in OVE26 diabetic mouse model podocyte by microarray assay while normal FVB mouse podocyte set as control.
Project description:The aim of the present study was to identify novel DNA methylation markers in bladder cancer (BCa) through genome-wide profiling of bladder cancer cell lines and subsequent MSP screening in urine samples. Experimental Design: MBD methylCap/seq was carried out to screen differentially methylated CpG islands using two BCa cell lines (5637 and T24) and two normal bladder mucosa (BM) samples. The top one hundred most hypermethylated targets were screened using Methylation Specific PCR (MSP) in small and big cohort of urine samples from BCa patients and normal controls. The diagnostic performance of the gene panel was further evaluated in different clinical scenarios. Results: In total, 1,627 gene promoter regions hypermethylated in BCa cell line were identified in genomic level methylation profiling. The followed screening procedure in clinical urine sample generated eight genes (VAX1, KCNV1, ECEL1, TMEM26, TAL1, PROX1, SLC6A20, and LMX1A) capable of differentiating BCa from normal control. Subsequent validation in a large sample size enabled the optimisation of 5 methylation targets (VAX1, KCNV1, TAL1, PPOX1 and CFTR) for BCa diagnosis with sensitivity and specificity of 86.32% and 87.13%, respectively. In addition, VAX1 and LMX1A methylation could predict the tumour recurrence. Conclusions: Tumor specific biomarkers of BCa could be established by first performing genome level methylation profiling with cell lines and then screening the potential targets in urine samples. The panel of methylated genes identified was promising for the early non-invasive detection and surveillance of BCa. MBD methylCap/seq was carried out to screen differentially methylated CpG islands using two BCa cell lines (5637 and T24), and two normal bladder tissue mix as control.
Project description:The aim of the present study was to identify novel DNA methylation markers in bladder cancer (BCa) through genome-wide profiling of bladder cancer cell lines and subsequent MSP screening in urine samples. Experimental Design: MBD methylCap/seq was carried out to screen differentially methylated CpG islands using two BCa cell lines (5637 and T24) and two normal bladder mucosa (BM) samples. The top one hundred most hypermethylated targets were screened using Methylation Specific PCR (MSP) in small and big cohort of urine samples from BCa patients and normal controls. The diagnostic performance of the gene panel was further evaluated in different clinical scenarios. Results: In total, 1,627 gene promoter regions hypermethylated in BCa cell line were identified in genomic level methylation profiling. The followed screening procedure in clinical urine sample generated eight genes (VAX1, KCNV1, ECEL1, TMEM26, TAL1, PROX1, SLC6A20, and LMX1A) capable of differentiating BCa from normal control. Subsequent validation in a large sample size enabled the optimisation of 5 methylation targets (VAX1, KCNV1, TAL1, PPOX1 and CFTR) for BCa diagnosis with sensitivity and specificity of 86.32% and 87.13%, respectively. In addition, VAX1 and LMX1A methylation could predict the tumour recurrence. Conclusions: Tumor specific biomarkers of BCa could be established by first performing genome level methylation profiling with cell lines and then screening the potential targets in urine samples. The panel of methylated genes identified was promising for the early non-invasive detection and surveillance of BCa.
Project description:Urine represents an ideal source of clinically relevant biomarkers since it contains a large number of proteins and low molecular weight peptides. Characterization of the normal urinary proteome and peptidome can serve as a reference for disease and can aid in biomarker discovery. Proteomic and peptidomic analysis of urine can also provide insight into normal physiology and disease pathology, especially for urogenital diseases. We developed an integrated proteomic and peptidomic analytical protocol for normal urine. We employed ultrafiltration to separate protein and peptide fractions, which were analyzed separately using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) on the Q-Exactive mass spectrometer. By analyzing six urines from healthy individuals, we identified 1754 proteins by proteomic analysis and 4543 endogenous peptides, arising from 566 proteins by peptidomic analysis. Overall, we identified 2091 non-redundant proteins by this integrated approach. In silico protease activity analysis indicated that metalloproteases are predominantly involved in the generation of the endogenous peptide signature. In addition, a number of proteins that were detected in normal urine have previously been implicated in various urological malignancies, including bladder cancer and renal cell carcinoma. Thus, this study can provide a reference for assessing alterations in cancer-specific biomarkers.
Project description:Urine is an ideal material to study and examine the bladder cancer (BCa) biomarkers, whereas exploration to the corresponding protein candidates is confronting technique challenges. Herein, we proposed a comprehensive strategy of searching the urine proteins related to BCa. The strategy consists of three core combinations, screening the candidates in the secreted proteins derived from the BCa cell lines and verifying them in the patient urines, defining the differential proteins through two-dimensional electrophoresis (2DE) and isobaric tags for relative and absolute quantitation (iTRAQ), and implementing quantitative analysis in profiling and targeting proteomics. The differential proteins were globally and quantitatively determined between the two typical cell lines of BCa, T24 and 5637, and the immortalized normal uroepithelium cell line, SV-HUC-1, while the BCa related proteins were verified between the relatively normal and patient urines. With proteomic survey combined with 2DE and iTRAQ, a total of 724 secreted proteins were identified as the BCa related proteins. With label-free quantitative proteomics, the 96 candidates were detected in the pooled urine samples. Furthermore, the multiple reaction monitoring (MRM)-based quantification was adapted to verify these urine proteins in individual urines that were collected from 23 BCa patients and 24 relative normal people, and resulted in the 10 urine proteins with significant abundance differences between the two groups. Of the potential indicators for BCa, analysis of receiver operating characteristics (ROC) revealed the combination of complement component 3 (CO3) and lactose dehydrogenase B (LDHB) was more sensitive and efficient to distinguish healthy and disease urine. The discovery of the indicative proteins of BCa through our strategy has thus paved an avenue to further validation of BCa biomarkers in urine.
Project description:Podocytes form filtration barrier through foot process around glomerualar basement membrane and selectively permit permeability of molecular smaller than albumin. Diabetes can cause podocyte pathological changes leading to high urine albumin level. Diabetic mouse model OVE26 has extremly high urine albumin and previously studies indicated its podocyte damaged. Here we try to find the key genes change in OVE26 diabetic mouse model podocyte by microarray assay while normal FVB mouse podocyte set as control. Podocyte eGFP transgenic mice were made on FVB background and crossbred to OVE26 diabetic model. Glomeruli isolated from OVE-GFP mice were digested by trypsin into signal cell. Podocytes with GFP were sorting out by FACS.