Circular RNAs and Their Linear Transcripts as Diagnostic and Prognostic Tissue Biomarkers in Prostate Cancer after Prostatectomy in Combination with Clinicopathological Factors.

As new biomarkers, circular RNAs (circRNAs) have been largely unexplored in prostate cancer (PCa). Using an integrative approach, we aimed to evaluate the potential of circRNAs and their linear transcripts (linRNAs) to act as (i) diagnostic biomarkers for differentiation between normal and tumor tissue and (ii) prognostic biomarkers for the prediction of biochemical recurrence (BCR) after radical prostatectomy. In a first step, eight circRNAs (circATXN10, circCRIM1, circCSNK1G3, circGUCY1A2, circLPP, circNEAT1, circRHOBTB3, and circSTIL) were identified as differentially expressed via a genome-wide circRNA-based microarray analysis of six PCa samples. Additional bioinformatics and literature data were applied for this selection process. In total, 115 malignant PCa and 79 adjacent normal tissue samples were examined using robust RT-qPCR assays specifically established for the circRNAs and their linear counterparts. Their diagnostic and prognostic potential was evaluated using receiver operating characteristic curves, Cox regressions, decision curve analyses, and C-statistic calculations of prognostic indices. The combination of circATXN10 and linSTIL showed a high discriminative ability between malignant and adjacent normal tissue PCa. The combination of linGUCY1A2, linNEAT1, and linSTIL proved to be the best predictive RNA-signature for BCR. The combination of this RNA signature with five established reference models based on only clinicopathological factors resulted in an improved predictive accuracy for BCR in these models. This is an encouraging study for PCa to evaluate circRNAs and their linRNAs in an integrative approach, and the results showed their clinical potential in combination with standard clinicopathological variables.

Instability of circular RNAs in clinical tissue samples impairs their reliable expression analysis using RT-qPCR: from the myth of their advantage as biomarkers to reality.

Background: Circular RNAs (circRNAs) are a new class of RNAs with medical significance. Compared to that of linear mRNA transcripts, the stability of circRNAs against degradation owing to their circular structure is considered advantageous for their use as biomarkers. As systematic studies on the stability of circRNAs depending on the RNA integrity, determined as RNA integrity number (RIN), in clinical tissue samples are lacking, we have investigated this aspect in the present study under model and clinical conditions. Methods: Total RNA isolated from kidney cancer tissue and cell lines (A-498 and HEK-293) with different RIN after thermal degradation was used in model experiments. Further, RNA isolated from kidney cancer and prostate cancer tissue collected under routine surgical conditions, representing clinical samples with RIN ranging from 2 to 9, were examined. Quantitative real-time reverse-transcription polymerase chain reaction (RT-qPCR) analysis of several circRNAs (circEGLN3, circRHOBTB3, circCSNK1G3, circRNA4, and circRNA9), their corresponding linear counterparts, tissue-specific reference genes, and three microRNAs (as controls) was performed. The quantification cycles were converted into relative quantities and normalized to the expression of specific reference genes for the corresponding tissue. The effect of RIN on the expression of different RNA entities was determined using linear regression analysis, and clinical samples were classified into two groups based on RIN greater or lesser than 6. Results: The results of model experiments and clinical sample analyses showed that all relative circRNA expression gradually decreased with reduction in RIN values. The adverse effect of RIN was partially compensated after normalizing the data and limiting the samples to only those with RIN values > 6. Conclusions: Our results suggested that circRNAs are not stable in clinical tissue samples, but are subjected to degradative processes similar to mRNAs. This has not been investigated extensively in circRNA expression studies, and hence must be considered in future for obtaining reliable circRNA expression data. This can be achieved by applying the principles commonly used in mRNA expression studies.

Limited utility of qPCR-based detection of tumor-specific circulating mRNAs in whole blood from clear cell renal cell carcinoma patients.

BACKGROUND: RNA sequencing data is providing abundant information about the levels of dysregulation of genes in various tumors. These data, as well as data based on older microarray technologies have enabled the identification of many genes which are upregulated in clear cell renal cell carcinoma (ccRCC) compared to matched normal tissue. Here we use RNA sequencing data in order to construct a panel of highly overexpressed genes in ccRCC so as to evaluate their RNA levels in whole blood and determine any diagnostic potential of these levels for renal cell carcinoma patients. METHODS: A bioinformatics analysis with Python was performed using TCGA, GEO and other databases to identify genes which are upregulated in ccRCC while being absent in the blood of healthy individuals. Quantitative Real Time PCR (RT-qPCR) was subsequently used to measure the levels of candidate genes in whole blood (PAX gene) of 16 ccRCC patients versus 11 healthy individuals. PCR results were processed in qBase and GraphPadPrism and statistics was done with Mann-Whitney U test. RESULTS: While most analyzed genes were either undetectable or did not show any dysregulated expression, two genes, CDK18 and CCND1, were paradoxically downregulated in the blood of ccRCC patients compared to healthy controls. Furthermore, LOX showed a tendency towards upregulation in metastatic ccRCC samples compared to non-metastatic. CONCLUSIONS: This analysis illustrates the difficulty of detecting tumor regulated genes in blood and the possible influence of interference from expression in blood cells even for genes conditionally absent in normal blood. Testing in plasma samples indicated that tumor specific mRNAs were not detectable. While CDK18, CCND1 and LOX mRNAs might carry biomarker potential, this would require validation in an independent, larger patient cohort.

A Novel Predictor Tool of Biochemical Recurrence after Radical Prostatectomy Based on a Five-MicroRNA Tissue Signature.

Within five to ten years after radical prostatectomy (RP), approximately 15-34% of prostate cancer (PCa) patients experience biochemical recurrence (BCR), which is defined as recurrence of serum levels of prostate-specific antigen >0.2 µg/L, indicating probable cancer recurrence. Models using clinicopathological variables for predicting this risk for patients lack accuracy. There is hope that new molecular biomarkers, like microRNAs (miRNAs), could be potential candidates to improve risk prediction. Therefore, we evaluated the BCR prognostic capability of 20 miRNAs, which were selected by a systematic literature review. MiRNA expressions were measured in formalin-fixed, paraffin-embedded (FFPE) tissue RP samples of 206 PCa patients by RT-qPCR. Univariate and multivariate Cox regression analyses were performed, to assess the independent prognostic potential of miRNAs. Internal validation was performed, using bootstrapping and the split-sample method. Five miRNAs (miR-30c-5p/31-5p/141-3p/148a-3p/miR-221-3p) were finally validated as independent prognostic biomarkers. Their prognostic ability and accuracy were evaluated using C-statistics of the obtained prognostic indices in the Cox regression, time-dependent receiver-operating characteristics, and decision curve analyses. Models of miRNAs, combined with relevant clinicopathological factors, were built. The five-miRNA-panel outperformed clinically established BCR scoring systems, while their combination significantly improved predictive power, based on clinicopathological factors alone. We conclude that this miRNA-based-predictor panel will be worth to be including in future studies.

Circular RNAs in Clear Cell Renal Cell Carcinoma: Their Microarray-Based Identification, Analytical Validation, and Potential Use in a Clinico-Genomic Model to Improve Prognostic Accuracy.

Circular RNAs (circRNAs) may act as novel cancer biomarkers. However, a genome-wide evaluation of circRNAs in clear cell renal cell carcinoma (ccRCC) has yet to be conducted. Therefore, the objective of this study was to identify and validate circRNAs in ccRCC tissue with a focus to evaluate their potential as prognostic biomarkers. A genome-wide identification of circRNAs in total RNA extracted from ccRCC tissue samples was performed using microarray analysis. Three relevant differentially expressed circRNAs were selected (circEGLN3, circNOX4, and circRHOBTB3), their circular nature was experimentally confirmed, and their expression-along with that of their linear counterparts-was measured in 99 malignant and 85 adjacent normal tissue samples using specifically established RT-qPCR assays. The capacity of circRNAs to discriminate between malignant and adjacent normal tissue samples and their prognostic potential (with the endpoints cancer-specific, recurrence-free, and overall survival) after surgery were estimated by C-statistics, Kaplan-Meier method, univariate and multivariate Cox regression analysis, decision curve analysis, and Akaike and Bayesian information criteria. CircEGLN3 discriminated malignant from normal tissue with 97% accuracy. We generated a prognostic for the three endpoints by multivariate Cox regression analysis that included circEGLN3, circRHOBT3 and linRHOBTB3. The predictive outcome accuracy of the clinical models based on clinicopathological factors was improved in combination with this circRNA-based signature. Bootstrapping as well as Akaike and Bayesian information criteria confirmed the statistical significance and robustness of the combined models. Limitations of this study include its retrospective nature and the lack of external validation. The study demonstrated the promising potential of circRNAs as diagnostic and particularly prognostic biomarkers in ccRCC patients.

miR-9-5p in Nephrectomy Specimens is a Potential Predictor of Primary Resistance to First-Line Treatment with Tyrosine Kinase Inhibitors in Patients with Metastatic Renal Cell Carcinoma.

Approximately 20⁻30% of patients with metastatic renal cell carcinoma (mRCC) in first-line treatment with tyrosine kinase inhibitors (TKIs) do not respond due to primary resistance to this drug. At present, suitable robust biomarkers for prediction of a response are not available. Therefore, the aim of this study was to evaluate a panel of microRNAs (miRNAs) in nephrectomy specimens for use as predictive biomarkers for TKI resistance. Archived formalin-fixed, paraffin embedded nephrectomy samples from 60 mRCC patients treated with first-line TKIs (sunitinib, n = 51; pazopanib, n = 6; sorafenib, n = 3) were categorized into responders and non-responders. Using the standard Response Evaluation Criteria in Solid Tumors, patients with progressive disease within 3 months after the start of treatment with TKI were considered as non-responders and those patients with stable disease and complete or partial response under the TKI treatment for at least 6 months as responders. Based on a miRNA microarray expression profile in the two stratified groups of patients, seven differentially expressed miRNAs were validated using droplet digital reverse-transcription quantitative real-time polymerase chain reaction (RT-qPCR) assays in the two groups. Receiver operating characteristic curve analysis and binary logistic regression of response prediction were performed. MiR-9-5p and miR-489-3p were able to discriminate between the two groups. MiR-9-5p, as the most significant miRNA, improved the correct prediction of primary resistance against TKIs in comparison to that of conventional clinicopathological variables. The results of the decision curve analyses, Kaplan-Meier analyses and Cox regression analyses confirmed the potential of miR-9-5p in the prediction of response to TKIs and the prediction of progression-free survival after the initiation of TKI treatment.

Tissue-Based MicroRNAs as Predictors of Biochemical Recurrence after Radical Prostatectomy: What Can We Learn from Past Studies?

With the increasing understanding of the molecular mechanism of the microRNAs (miRNAs) in prostate cancer (PCa), the predictive potential of miRNAs has received more attention by clinicians and laboratory scientists. Compared with the traditional prognostic tools based on clinicopathological variables, including the prostate-specific antigen, miRNAs may be helpful novel molecular biomarkers of biochemical recurrence for a more accurate risk stratification of PCa patients after radical prostatectomy and may contribute to personalized treatment. Tissue samples from prostatectomy specimens are easily available for miRNA isolation. Numerous studies from different countries have investigated the role of tissue-miRNAs as independent predictors of disease recurrence, either alone or in combination with other clinicopathological factors. For this purpose, a PubMed search was performed for articles published between 2008 and 2017. We compiled a profile of dysregulated miRNAs as potential predictors of biochemical recurrence and discussed their current clinical relevance. Because of differences in analytics, insufficient power and the heterogeneity of studies, and different statistical evaluation methods, limited consistency in results was obvious. Prospective multi-institutional studies with larger sample sizes, harmonized analytics, well-structured external validations, and reasonable study designs are necessary to assess the real prognostic information of miRNAs, in combination with conventional clinicopathological factors, as predictors of biochemical recurrence.

Diagnostic and prognostic potential of circulating cell-free genomic and mitochondrial DNA fragments in clear cell renal cell carcinoma patients.

BACKGROUND: There is inconsistent information about the clinical usefulness of circulating cell-free DNA (cfDNA) in plasma from clear cell renal cell cancer (RCC) patients. This is attributed to preanalytical, analytical, and clinical factors that were considered as far as possible in this study. METHODS: cfDNA was extracted from EDTA plasma of healthy people (n=40), non-metastatic (n=145) and metastatic (n=84) RCC patients using the QIAamp Circulating Nucleic Acid Kit. Genomic and mitochondrial cfDNA concentrations were determined using qPCR of different cfDNA fragments (67-306bp). Their diagnostic and prognostic potential was estimated using receiver operating characteristics (ROC) and Cox regression analyses. RESULTS: The 67bp and 180bp genomic cfDNA fragments did not differ between the three study groups while the 306bp fragment was lower in RCC patients than in controls. The mitochondrial cfDNA was higher in metastatic than in non-metastatic patients and controls. The cfDNA integrity indices decreased from controls to metastatic patients. Models built by logistic regression and Cox regression resulted in area under the ROC curves >0.75 and concordance indices of >0.800 in predicting recurrence-free survival and overall survival. CONCLUSION: The study suggests that combinations of cfDNA markers have promising diagnostic and prognostic potential in RCC patients and are worth for further validation in future prospective multicenter studies.

Piwi-interacting RNAs as novel prognostic markers in clear cell renal cell carcinomas.

BACKGROUND: Piwi-interacting RNAs (piRNAs) are small RNAs of 27-30 nucleotides mapping to transposons or clustering in repeat genomic regions. Preliminary studies suggest an important role in cancerogenesis. This study is the first one investigating their prognostic impact in clear cell renal cell cancer (ccRCC) patients. METHODS: Three piRNAs (piR-30924, piR-57125, and piR-38756) selected on the basis of initial piRNA microarray analyses were determined using RT-qPCR in non-metastatic (n = 76) and metastatic (n = 30) ccRCC tissue at the time of nephrectomy in comparison to normal renal tissue (n = 77) and tissue from distant ccRCC metastases (n = 13). Primary clinical end points were recurrence-free and overall survival. RESULTS: piR-57125 showed lower expression in metastatic than in non-metastatic tumors, whereas the expression of piR-30924 and piR-38756 increased in metastatic tumors. The higher expression of piR-30924 and piR-38756 as well as the lower expression of piR-57125 in metastatic primary tumors were significantly associated with tumor recurrence and overall survival. Multivariate Cox regression analyses revealed both piR-30924 and piR-57125 as independent prognostic predictors. This impact was even more pronounced in non-metastatic patients. CONCLUSIONS: This study demonstrates that the expression levels of these piRNAs in primary non-metastatic and metastatic ccRCC tissue can serve as potential prognostic biomarkers in combination with clinicopathological factors.

Urinary miR-183 and miR-205 do not surpass PCA3 in urine as predictive markers for prostate biopsy outcome despite their highly dysregulated expression in prostate cancer tissue.

BACKGROUND: MicroRNAs (miRNAs) have shown to be promising novel biomarkers in various cancers. We aimed to translate the results of an own previous tissue-based miRNA profile of prostate carcinoma (PCa) with upregulated miR-183 and downregulated miR-205 into a urine-based testing procedure for diagnosis of PCa. METHODS: Urine sediments were prepared from urine samples collected after a standardized digital-rectal examination (DRE) of patients undergoing prostate biopsy with PSA (prostate-specific antigen) values <20 µg/L in consecutive order. According to the sample-size calculation (α=0.05, power=0.95), 38 patients each with PCa and without PCa were randomly enrolled in this study. PCA3 (prostate cancer associated 3) in urine as Food and Drug Administration-approved assay was determined as reference standard for comparison. The miRNAs were measured by RT-qPCR using TaqMan assays and normalized using different approaches. RESULTS: Both miRNAs were correlated to the mRNA PSA concentrations in the sediments indicating a relationship to the released prostate cells after DRE. However, they had no discriminating capacity between patients with and without PCa. In contrast, PCA3 clearly differentiated between these two patients groups. There was also no significant correlation between miRNAs and standard clinicopathologic variables like Gleason score and serum PSA. CONCLUSIONS: The data of our study show that miR-183 and miR-205 failed to detect early and aggressive PCa despite their highly dysregulated expression in cancer tissue. Our results and the critical evaluation of the few data of other studies raise serious doubts concerning the capability of urinary miRNAs to replace or improve PCA3 as predictive marker for prostate biopsy outcome.

The antiapoptotic function of miR-96 in prostate cancer by inhibition of FOXO1.

microRNAs (miRNAs) are small molecules that regulate gene expression posttranscriptionally. In a previous study, we identified miR-96 to be upregulated in prostate cancer specimens in comparison to normal adjacent tissue and to be an independent marker of biochemical relapse in a multivariate prediction model. Therefore, we investigated the functional role of miR-96 in prostate carcinogenesis. LNCaP and DU145 prostate cancer cells were transiently transfected with miR-96 precursors and phenotypic changes were analyzed. The miR-96 increased proliferation and impaired apoptosis induced by camptothecine in these cells. In silico target prediction analysis identified FOXO1 as potential pro-apoptotic miR-96 target. miR-96 was able to bind to both bindings sites in the FOXO1 3' UTR in a luciferase reporter gene assay. Overexpression of miR-96 in LNCaP cells resulted in a reduced FOXO1 expression. Overexpression of FOXO1 induced a strong apoptotic phenotype that was partially rescued by coexpression of miR-96. RT-qPCR and immunohistochemistry of 69 prostate cancer specimens revealed a downregulation of FOXO1 and an inverse correlation of miR-96 and FOXO1 protein expression. In conclusion, we show that miR-96 can regulate apoptosis in prostate cancer, by inhibiting the FOXO1 transcription factor.

miRNA profiling identifies candidate mirnas for bladder cancer diagnosis and clinical outcome.

Bladder cancer is a common cancer in the Western world. The current prognosticators such as tumor grade, stage, size, and multifocality do not accurately reflect the clinical outcome. It is of clinical interest to identify biomarkers that could improve diagnostic and/or prognostic predictions. The objectives of this study were to identify deregulated miRNAs in bladder cancer samples and evaluate their potential as diagnostic and prognostic biomarkers. We screened 723 miRNAs by microarray and selected a subset of 15 distinctively deregulated miRNAs for further validation by real-time quantitative RT-(q)PCR. Seven miRNAs (miR-20a, miR-106b, miR-130b, miR-141, miR-200a, miR-200a*, and miR-205) were found to be up-regulated and eight miRNAs (miR-100, miR-125b, miR-130a, miR-139-5p, miR-145*, miR-199a-3p, miR-214, and miR-222) were found to be down-regulated in malignant bladder tissue samples compared to healthy tissue. Four miRNAs that have already been described in the literature (miR-141, miR-199a-3p, miR-205, and miR-214) were significantly differentially expressed between nonmuscle-invasive and muscle-invasive bladder cancer. Furthermore, real-time RT-qPCR of all miRNAs provided high overall correct classification (>75%) of bladder cancer diagnosis. Two miRNAs (miR-141 and miR-205) were associated with overall survival time. The verification of tumor-specific miRNA expression profile, together with the observed association of miR-141 and miR-205 expression with overall survival, underline the potential of miRNAs to function as diagnostic and/or prognostic markers of bladder cancer.

Identification of microRNAs in blood and urine as tumour markers for the detection of urinary bladder cancer.

Since differential expression of microRNAs (miRNAs) has been found to be highly associated with several types of cancer, the goal of the present study was to identify an miRNA fingerprint as a non­invasive diagnostic tool to detect urinary bladder cancer using the easily accessible samples of whole blood and urine. Blood and urine samples from 4 controls and from patients suffering from superficial and invasive bladder cancer were analyzed using miRNA microarray consisting of 754 human miRNAs from the Sanger database v14. Using RT­qPCR technique, 6 of the differentially expressed miRNAs were validated in the controls (20 blood, 19 urine samples) and patients with superficial (18 blood, 16 urine samples) or invasive (20 blood and urine samples each) tumours. Three blood miRNAs (miR­26b­5p, miR­144­5p, miR­374­5p) were found to be significantly upregulated in invasive bladder tumour patients (P<0.05) when compared to the control group. The expression of 2 miRNAs (miR­618, miR­1255b­5p) in the urine of patients with invasive tumours was significantly (P<0.05) increased in comparison to the control group. Blood miR­26b­5p detected the presence of invasive bladder tumours with 94% specificity and 65% sensitivity. The urine miR­1255b­5p reached 68% specificity and 85% sensitivity in the diagnosis of invasive tumours. This pilot study represents the first characterization of an miRNA profile for urinary bladder tumours in whole blood samples. In addition, it was shown that invasive bladder tumours could be identified by differentially expressed urine miRNAs. Further studies are needed to test the clinical usefulness for bladder cancer detection and surveillance.

Diagnostic and prognostic potential of differentially expressed miRNAs between metastatic and non-metastatic renal cell carcinoma at the time of nephrectomy.

BACKGROUND: MicroRNAs are promising diagnostic and prognostic biomarkers in oncology. We aimed to evaluate the prognostic potential of selected microRNAs in primary clear cell renal cell carcinomas (ccRCC) as predictors of tumor recurrence after radical nephrectomy. METHODS: miR-122, miR-141, miR-155, miR-184, miR-200c, miR-210, miR-224, and miR-514, validated as differentially expressed in a previous study, were measured by RT-PCR in matched malignant and non-malignant tumor samples after nephrectomy from 111 patients (89 without, 22 with metastases) and clinicopathological and outcome data were collected. Non-parametric statistical tests, receiver-operating characteristics, Kaplan-Meier-, and univariate as well as multivariate Cox regression analyses were performed. RESULTS: Downregulation of miR-141/-184/-200c/-514 and upregulation of miR-122/-155/-210/-224 were not different between samples of non-metastatic and metastatic tumors except for miR-122 and miR-514. miR-514 was further downregulated in metastatic compared with non-metastatic tumors while the upregulation of miR-122 was significantly reduced in metastatic carcinomas. All miRNAs were suitable to discriminate malignant from non-malignant tissue. miR-122 and miR-514 were significantly related to the recurrence risk but only miR-514 provided independent prognostic information in the final model including relevant clinicopathological variables. CONCLUSIONS: MiR-122 and miR-514 play a role in tumor recurrence after nephrectomy. Expression of miR-514 was particularly downregulated in primary metastatic tumor and those that recur and might be a suitable adjunct marker for predicting tumor recurrence.

Identification of metastamirs as metastasis-associated microRNAs in clear cell renal cell carcinomas.

MicroRNAs (miRNAs) play a pivotal role in cancerogenesis and cancer progression, but their specific role in the metastasis of clear cell renal cell carcinomas (ccRCC) is still limited. Based on microRNA microarray analyses from normal and cancerous samples of ccRCC specimens and from bone metastases of ccRCC patients, we identified a set of 57 differentially expressed microRNAs between these three sample groups of ccRCC. A selected panel of 33 miRNAs was subsequently validated by RT-qPCR on total 57 samples. Then, 30 of the 33 examined miRNAs were confirmed to be deregulated. A stepwise down-regulation of miRNA expression from normal, over primary tumor to metastatic tissue samples, was found to be typical. A total of 23 miRNAs (miR-10b/-19a/-19b/-20a/-29a/-29b/-29c/-100/-101/-126/-127/-130/-141/-143/-145/-148a/-192/-194/-200c/-210/-215/-370/-514) were down-regulated in metastatic tissue samples compared with normal tissue. This down-regulated expression in metastatic tissue in comparison with primary tumor tissue was also present in 21 miRNAs. In cell culture experiments with 5-aza-2'-deoxycytidine and trichostatin A, epigenetic modifications were shown as one reason of this down-regulation. The altered miRNA profiles, comprising newly identified metastasis-associated miRNAs, termed metastamir and the predicted miRNA-target interactions together with the significant correlations of miRNAs that were either lost or newly appeared in the studied sample groups, afford a solid basis for further functional analyses of individual miRNAs in RCC metastatic progression.

Reference miRNAs for miRNAome analysis of urothelial carcinomas.

BACKGROUND/OBJECTIVE: Reverse transcription quantitative real-time PCR (RT-qPCR) is widely used in microRNA (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. 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 miRNAs for miRNA expression studies by RT-qPCR in urothelial carcinoma. METHODS: Candidate reference miRNAs were selected from 24 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-151-5p, 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-101, miR-125a-5p, miR-148b, and miR-151-5p) or three (miR-148b, miR-181b, and miR-874,) reference miRNAs is recommended for normalization.

Reference genes for the relative quantification of microRNAs in renal cell carcinomas and their metastases.

To obtain accurate results in miRNA expression changes between different sample sets using real-time quantitative polymerase chain reaction (RT-qPCR) analyses, normalization to reference genes that are stably expressed across the sample sets is generally used. A literature search of miRNA expression studies in renal cell carcinoma (RCC) proved that non-miRNAs such as small RNAs or mRNAs have most frequently been used without preceding validation of their suitability. In this study, the most stably expressed miRNAs were ascertained from microarray-based data of miRNA expression in nonmalignant and malignant samples from clear cell RCC and from corresponding distant RCC metastases using the geNorm and NormFinder algorithms. Validation experiments with RT-qPCR were performed for the four best-ranked miRNAs (miR-28, miR-103, miR-106a, miR-151) together with the small RNU6B, RNU44, and RNU48 mostly described in literature. miR-28, miR-103, miR-106a, and RNU48 were proved as the most stably expressed genes. miR-28 is recommended as normalizer if only a single reference gene can be used, while the combinations of miR-28 and miR-103 or of miR-28, miR-103, and miR-106a, respectively, are preferred. RNU6B most frequently used as normalizer in miRNA expression studies should be abandoned in order to avoid misleading results.

Fatty acid binding proteins (FABPs) in prostate, bladder and kidney cancer cell lines and the use of IL-FABP as survival predictor in patients with renal cell carcinoma.

BACKGROUND: Fatty acid binding proteins (FABP) play an important role in carcinogenesis. Modified FABP expression patterns were described for prostate, bladder and for renal cell carcinoma. Studies on metabolic relationships and interactions in permanent cell lines allow a deeper insight into molecular processes. The aim of this study is therefore a systematic overview on mRNA and protein expressions of seven FABPs in frequently used urological cell lines. METHODS: Nine cell lines of renal carcinomas, seven of urinary bladder carcinomas, and five of prostate carcinomas were investigated. Quantitative RT-qPCR and western blotting were used to determine different FABPs. In addition, 46 paired cancerous and noncancerous tissue samples from nephrectomy specimen with renal cell carcinomas were investigated regarding the ileum FABP mRNA expression level and associated with survival outcome. RESULTS: General characteristics of all urological carcinoma cell lines were the expression of E-and IL-FABP on mRNA and protein level, while the expressions differed between the cell lines. The protein expression was not always congruent with the mRNA expression. Renal cell carcinoma cell lines showed expressions of L-, H- and B-FABP mRNA in addition to the general FABP expression in five out of the eight investigated cell lines. In bladder cancer cell lines, we additionally found the expression of A-FABP mRNA in six cell lines, while H-FABP was present only in three cell lines. In prostate cancer cell lines, a strong reduction of A- and E- FABP mRNA was observed. The expression of B-FABP mRNA and protein was observed only in the 22 RV-1 cells. IL-FABP mRNA was over-expressed in renal tumour tissue. The IL-FABP ratio was identified as an independent indicator of survival outcome. CONCLUSIONS: Distinctly different FABP expression patterns were observed not only between the cell lines derived from the three cancer types, but also between the cell lines from the same cancer. The FABP patterns in the cell lines do not always reflect the real situation in the tumours. These facts have to be considered in functional studies concerning the different FABPs.

miRNAs can predict prostate cancer biochemical relapse and are involved in tumor progression.

Prostate cancer is the leading cancer diagnosed and the second most common cause of cancer related death in the western world. For prognostic monitoring after prostatectomy, recurrent increase of prostate-specific antigen (PSA), the so-called PSA or biochemical relapse remains the leading biomarker. There is currently no biomarker that can accurately predict the risk of relapse at the time of surgery. We analyzed formalin-fixed and paraffin-embedded tissue samples from 52 primary prostate cancers and normal adjacent tissues obtained after radical prostatectomy. Patients were grouped into two categories: i) patients with early biochemical relapse (<1 year after radical prostatectomy) and ii) patients with late or no biochemical relapse (>2 years after surgery). Multiplex real-time quantitative polymerase chain reaction (RT-qPCR) analysis was performed to identify a miRNA signature that can predict relapse. Results were validated by quantitative RT-PCR analysis. We identified 63 miRNAs that were differentially expressed among the same categories of patients, of whom 35 miRNAs were up-regulated and 28 were down-regulated. Literature search shows that many of these miRNAs have an established prognostic significance in other cancers and can be actively involved in tumor progression. Target prediction analysis showed that predicted targets of these miRNAs could be involved in biological processes and pathways that enhance tumor progression. We experimentally validated the role of one of the dysregulated miRNAs (miR-10b) in relapse using proliferation and wound-healing assay. miRNAs can be reliable predictive markers for biochemical relapse of prostate cancer at the time of radical prostatectomy. This should have significant impact on patient managing plans.

Suitable reference genes for relative quantification of miRNA expression in prostate cancer.

Real time quantitative PCR (qPCR) is the method of choice for miRNA expression studies. For relative quantification of miRNAs, normalization to proper reference genes is mandatory. Currently, no validated reference genes for miRNA qPCR in prostate cancer are available. In this study, the expression of four putative reference genes (hsa-miR-16, hsa-miR-130b, RNU6-2, SNORD7) was examined with regard to their use as normalizer. After SNORD7 was already shown an inappropriate reference gene in preliminary experiments using total RNA pools, we studied the expression of the putative reference genes in tissue and normal adjacent tissue sample pairs from 76 men with untreated prostate carcinoma collected after radical prostatectomy. hsa-miR-130b and RNU6-2 showed no significantly different expression between the matched malignant and non-malignant tissue samples, whereas hsa-miR-16 was significantly underexpressed in malignant tissue. Softwares geNorm and Normfinder predicted hsa- miR-130b and the geometric mean of hsa-miR-130b and RNU6-2 as the most stable reference genes. Normalization of the four miRNAs hsa-miR-96, hsa- miR-125b, hsa-miR-205, and hsa-miR-375, which were previously shown to be regulated, shows that normalization to hsa-mir-16 can lead to biased results. We recommend using hsa-miR-130b or the geometric mean of hsa-miR-130b and small RNA RNU6-2 for normalization in miRNA expression studies of prostate cancer.