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Bladder cancer: analysis of miRNA-mRNA interactions

ABSTRACT: Background: MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression. Their abilyty to affect multiple gene pathways by targeting to various mRNAs makes them to an interesting class of regulators. The interplay between miRNA and mRNA has been proposed as an important process in cancer development and progression. In this study, we analyzed miRNA -mRNA interactions in bladder urothelial carcinoma. Methodology/Principal Findings: We have developed a new algorithm which is capable of identifying altered miRNA-mRNA regulation between tissues samples without preexisting stratification of groups. Microarray expression profiling of both miRNA and mRNA from the same sample were performed using a collective of 24 urothelial carcinoma and normal bladder tissue samples. Using our approach, normal and tumor tissue samples as well as different stages of tumor progression were successfully stratified. Also, we were able to analyzed individual miRNA-mRNA interactions from each patient, focusing on different miRNA families. Conclusions: Just recently, the need for tools that allow an integrative analysis of microRNA and mRNA expression data has been addressed. With this study, we provide an algorithm that considers the special nature of miRNA induced regulation and shows good specificities and sensitivities when applied to bladder cancer expression data.

ORGANISM(S): Homo sapiens

PROVIDER: GSE40355 | GEO | 2013/08/24

SECONDARY ACCESSION(S): PRJNA173681

REPOSITORIES: GEO

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Project description:Background/Objective: Quantitative real-time PCR (RT-qPCR) is widely used in miRNA expression studies on cancer. To compensate for the analytical variability produced by the multiple steps of the method, relative quantification of the measured miRNAs is required, which is based on normalization to endogenous reference genes. A literature search in PubMed revealed that no study has been performed so far on reference miRNAs for normalization of miRNA expression in urothelial carcinoma. The aim of this study was to identify suitable reference genes for miRNA expression studies by RT-qPCR in urothelial carcinoma. Methods: Candidate reference miRNAs were selected from 24 papillary urothelial carcinoma and normal bladder tissue samples by miRNA microarrays. The usefulness of these candidate reference miRNAs together with the commonly for normalization purposes used small nuclear RNAs RNU6B, RNU48, and Z30 were thereafter validated by RT-qPCR in 58 tissue samples and analyzed by the algorithms geNorm, NormFinder, and BestKeeper. Principal Findings: Based on the miRNA microarray data, a total of 16 miRNAs were identified as putative reference genes. After validation by RT-qPCR, miR-101, miR-125a-5p, miR-148b, miR-151-3p, miR-151-5p, miR-181a, miR-181b miR-29c, miR-324-3p, miR-424, miR-874, RNU6B, RNU48, and Z30 were used for geNorm, NormFinder, and BestKeeper analyses that gave different combinations of recommended reference genes for normalization. Conclusions: The present study provided the first systematic analysis for identifying suitable reference miRNAs for miRNA expression studies of urothelial carcinoma by RT-qPCR. Different combinations of reference genes resulted in reliable expression data for both strongly and less strongly altered miRNAs. Notably, RNU6B, which is the most frequently used reference gene for miRNA studies, gave inaccurate normalization. The combination of four (miR-125a-5p, miR-148b, miR-151-3p, and miR-151-5p) or three (miR-148b, miR-874, miR-181b) miRNA reference genes is recommended for normalization. Candidate reference miRNAs (for RT-qPCR analysis) were selected from 24 papillary urothelial carcinoma and normal bladder tissue samples by miRNA microarrays.

Project description:Expression profiling by arrays Urothelial carcinoma (UC) can arise at any location along the urothelial tract, including the urethra, bladder, ureter or renal pelvis. Although tumors arising in these various locations demonstrate similar morphology, it is unclear whether the gene expression profiles are similar in the upper tract (ureter and renal pelvis) or in the lower tract (bladder and urethra) carcinomas, especially given their different embryologic origins. As differences may facilitate potentially different screening and treatment modalities, we sought to examine the relationship between urothelial carcinoma of the renal pelvis (rUC) and urothelial carcinoma of the bladder (bUC). Fresh tumor tissue was collected from patients with bUC (n=10) and benign mucosa from the bladder (n=7) was collected from individuals undergoing resection for non-UC conditions for comparison. Gene expression profiles from these samples were determined using high-throughput Affymetrix gene expression microarray chips. Bioinformatic approaches were used to compare gene expression profiles of these samples and those of rUC (n= 14) and normal kidney (n=14) that were mostly used in our previous publication. Using unsupervised analytic approaches, rUC and bUC were indistinguishable. When supervised analytic approach was used, a very small number of potentially differentially expressed genes was identified; these differences were most likely to be limited to a single pathway - the chloride ion binding activity pathway -which was more frequently activated in rUC than in bUC. We found that the gene expression profiles of UCs from the upper and lower tract were extremely similar, suggesting that similar pathogenic mechanisms likely function in the development of these tumors. The differential expression of genes in the identified pathway may represent a potential new avenue for detection of upper tract tumors. Tissue samples with urothelial cell carcinoma from lower tract (bladder) as well as normal references were collected and the gene expression profiles were compared with gene expression profiles of samples in our previously published data set . No technical replicates.

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Bladder cancer: analysis of miRNA-mRNA interactions

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