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 is a non-invasive biofluid for the identification of biomarkers to detect disease. In particular extracellular vesicles (EVs) have gained increased interest as a biomarker source, because the molecular content is protected against degradation. Clinical implementation on a daily basis requires protocols that inevitably includes short-term storage of the clinical samples, especially when samples are collected at home. However, little is known about the effect of delayed processing on the urinary EVs and their proteome. In the current study, we evaluated two different storage protocols. First, urine stored at 4˚C without any preservative, and second, a protocol compatible with at-home collection, urine with 40 mM EDTA stored at room temperature. For both conditions it was determined whether storage for 0, 2, 4 and 8 days leads to a change in the global urinary EV proteome profile using proteomics based on data-independent acquisition mass spectrometry. We show that EDTA does not affect the global proteome. Remarkably, the EV proteome was stable in both urine stored up to a week at room temperature with EDTA and in urine stored at 4˚C. These findings open up biomarker studies in urine collected via self-sampling.
Project description:Discrepancies in blood sample collection and processing could have a significant impact on levels of peptides in the blood, thus sample quality control is critical for successful biomarker identification and validation. In this study, we analyzed the effects of several pre-analytical processing conditions, including different storage times and temperatures of blood or plasma samples and different centrifugation forces, on the levels of peptides in human plasma samples using ethylenediaminetetraacetic acid (EDTA) as an anticoagulant. Both time and temperature were identified as major factors for peptide variation.
Project description:Intra-individual stability of the urine miRNA transcriptome was examined by investigating longitudinal changes over time in a cohort of patients with localized prostate cancer. Using training and validation cohorts, urinary miRNA biomarkers are characterized and validated their utility to identify aggressive prostate cancer.
Project description:Intra-individual stability of the urine miRNA transcriptome was examined by investigating longitudinal changes over time in a cohort of patients with localized prostate cancer. Using training and validation cohorts, urinary miRNA biomarkers are characterized and validated their utility to identify aggressive prostate cancer.
Project description:Intra-individual stability of the urine miRNA transcriptome was examined by investigating longitudinal changes over time in a cohort of patients with localized prostate cancer. Using training and validation cohorts, urinary miRNA biomarkers are characterized and validated their utility to identify aggressive prostate cancer.
Project description:We performed single cell transcriptomic analysis on 17 urine samples obtained from five subjects at two different occasions using both spot and 24-hour urine collection. In addition, we used a combined spot urine samples of five healthy individuals as a control sample. We sequenced a total of 71,667 cells. After quality control and downstream analysis, we found that epithelial cells were the most common cell types in the urine. We were also able to identify most kidney cell types in the urine, such as podocyte, proximal, and collecting duct (CD), in addition to macrophages, monocytes and lymphocytes.
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.