Project description:Background: Renal cell carcinoma (RCC) accounts for about 2% of all cancers. Renal biopsy is the gold standard among the diagnostic tools, but it is invasive and not suitable for all patients. Therefore, new reliable and non-invasive biomarkers for ccRCC detection are required. Secretion of extracellular vesicles (EVs), containing RNA molecules that can be transferred between cells, seems to be a general characteristic of malignant transformation. Consistently, cancer-derived EVs are enriched in the blood, urine and various malignant effusions of cancer patients. Therefore, urinary samples can be a non-invasive approach for discovering diagnostic biomarkers. Methods: We enrolled 33 clear-cell RCC (ccRCC) patients and 22 healthy subjects (HS), age and sex-matched, for urine collection and extracellular vesicles isolation by differential centrifugation. Transcriptional profiles of urinary EVs from 12 patients with ccRCC and 11 HS were generated using the Illumina HumanHT-12 v4 BeadChip oligonucleotide arrays. Microarray analysis led to the identification of RNA that were then validated using RT-qPCR. Results: We showed for the first time that urinary exosomal shuttle RNA (esRNA) was significantly different in ccRCC patients compared to HS and we identified three EVs esRNA involved in the tumor biology that are potentially suitable as non-invasive biomarkers. GSTA1, CEBPA and PCBD1 RNA levels decreased in urinary EVs of patients compared to HS. After 1 month post-operation, the levels of RNA increased to reach the normal level. Conclusions: This study suggests, for the first time, the potential use of the RNA content of urinary EVs to provide a non-invasive first step to diagnose the ccRCC. Total RNA obtained from urinary extracellular vesicles isolated from ccRCC patients and healthy subjects.
Project description:Background: Renal cell carcinoma, which presents no significant clinical manifestations at early stages, is one of the few tumors with an increasing worldwide incidence. This malignancy cannot be directly detected by tumor markers in body fluid without information from imaging examinations. Approximately 30–40% of patients with kidney cancer exhibit distant metastasis at diagnosis, and their 5-year survival rate is much lower than that of patients with early-stage renal cell carcinoma. Thus, the early diagnosis of renal cell carcinoma is extremely important. The aim of this study was to investigate the utility of urinary exosomal long noncoding RNA (lncRNA) as a new potential diagnostic marker for renal cell carcinoma. Methods: Exosomes were isolated by ultracentrifugation from 50-ml urine samples from 10 patients with clear cell renal cell carcinoma and 10 matched healthy donors. Differentially expressed lncRNAs were analyzed by next-generation sequencing and further validated in kidney cancer cell lines, tissues and urinary exosomes. Then, we evaluated the sensitivity, specificity, clinical diagnostic value and stability of the selected lncRNAs. Results: The levels of lncRNAs NR_040448 and NR_033390 in urinary exosomes can likely indicate the presence of renal cell carcinoma. In addition, RNA protected by exosomes was stable enough to serve as a biomarker. Conclusion: Urinary exosomal lncRNA is a promising marker for the early diagnosis of renal cell carcinoma.
Project description:Translating the research capability and knowledge in cancer signaling into clinical settings has been slow and ineffective. Recently, extracellular vesicles (EVs) have emerged as a promising source for developing disease phosphoprotein markers to monitor disease status. This study focuses on the development of a robust data-independent acquisition (DIA) using mass spectrometry to profile urinary EV phosphoproteomics for renal cell cancer (RCC) grades differentiation. We examined gas-phase fractionated (GPF) library, direct DIA (library-free), forbidden zones, and several different windowing schemes. After the development of a DIA mass spectrometry method for EV phosphoproteomics, we applied the strategy to identify and quantify urinary EV phosphoproteomes from 57 individuals representing low-grade clear cell RCC, high-grade clear cell RCC, chronic kidney disease (CKD), and healthy control (HC) individuals. Urinary EVs were efficiently isolated by functional magnetic beads, and EV phosphopeptides were subsequently enriched by PolyMAC. We quantified 2,584 unique phosphosites and observed that multiple prominent cancer-related pathways, such as ErbB signaling, renal cell carcinoma, and regulation of actin cytoskeleton, were only upregulated in high-grade clear cell RCC. These results show that EV phosphoproteome analysis utilizing our optimized procedure of EV isolation, phosphopeptide enrichment, and DIA method provides a powerful tool for future clinical applications.
Project description:Background: Renal cell carcinoma (RCC) accounts for about 2% of all cancers. Renal biopsy is the gold standard among the diagnostic tools, but it is invasive and not suitable for all patients. Therefore, new reliable and non-invasive biomarkers for ccRCC detection are required. Secretion of extracellular vesicles (EVs), containing RNA molecules that can be transferred between cells, seems to be a general characteristic of malignant transformation. Consistently, cancer-derived EVs are enriched in the blood, urine and various malignant effusions of cancer patients. Therefore, urinary samples can be a non-invasive approach for discovering diagnostic biomarkers. Methods: We enrolled 33 clear-cell RCC (ccRCC) patients and 22 healthy subjects (HS), age and sex-matched, for urine collection and extracellular vesicles isolation by differential centrifugation. Transcriptional profiles of urinary EVs from 12 patients with ccRCC and 11 HS were generated using the Illumina HumanHT-12 v4 BeadChip oligonucleotide arrays. Microarray analysis led to the identification of RNA that were then validated using RT-qPCR. Results: We showed for the first time that urinary exosomal shuttle RNA (esRNA) was significantly different in ccRCC patients compared to HS and we identified three EVs esRNA involved in the tumor biology that are potentially suitable as non-invasive biomarkers. GSTA1, CEBPA and PCBD1 RNA levels decreased in urinary EVs of patients compared to HS. After 1 month post-operation, the levels of RNA increased to reach the normal level. Conclusions: This study suggests, for the first time, the potential use of the RNA content of urinary EVs to provide a non-invasive first step to diagnose the ccRCC.
Project description:The proteome of clinical tissue samples diagnosed with clear cell renal cell carcinoma (ccRCC) and papillary renal cell carcinoma (pRCC) were evaluated analyzed along with the dataset identifier PXD022018 to establish a potential discriminative biomarker panel of proteins for these tumors subtypes.
Project description:Extracellular vesicles isolated from the supernatant of luciferase-expressing renal cell carcinoma cell line (786-O luc) and its bone metastatic variant (786-O BM) which was established by in vivo selection method. Extracellular vesicles were isolated using MagCapture.
Project description:Long non-coding RNAs (lncRNA) play an important role in carcinogenesis, but knowledge of lncRNA expression in renal cell carcinoma is rudimental. We screened 32,183 lncRNA transcripts in malignant and adjacent normal renal tissue and determined dysregulation of approximately 4% of the lncRNA transcripts in clear cell renal cell carcinoma tissue. The distinct changes of lncRNA expression may be used to develop a non-invasive biomarker, because lncRNAs are detectable in bodily fluids. Microarray experiments were performed to determine the expression of 32,183 lncRNA transcripts belonging to 17,512 lncRNAs in 15 corresponding normal and malignant renal tissues
Project description:Bladder cancer is one of the most common cancers. Since prognosis ameliorates with early detection, it is a challenge to develop techniques that could replace or complement the current diagnosis protocols. The study of extracellular vesicles (EVs) that are present in urine samples has become an attractive alternative. The present study describes the mRNA content of vesicles isolated from voided urine samples within bladder cancer context. To discover a genetic signature of cancer, RNA associated to EVs was analyzed by microarray technique. Total RNA isolated from Extracellular Vesicles obtained from urine of bladder cancer patients was compared with RNA isolated from urinary vesicles of non-cancer patients.
Project description:Long non-coding RNAs (lncRNA) play an important role in carcinogenesis, but knowledge of lncRNA expression in renal cell carcinoma is rudimental. We screened 32,183 lncRNA transcripts in malignant and adjacent normal renal tissue and determined dysregulation of approximately 4% of the lncRNA transcripts in clear cell renal cell carcinoma tissue. The distinct changes of lncRNA expression may be used to develop a non-invasive biomarker, because lncRNAs are detectable in bodily fluids.