Monika Jung

Diagnostic and prognostic implications of microRNA profiling in prostate carcinoma

This study aimed to investigate the microRNA (miRNA) profile in prostate carcinoma tissue by microarray analysis and RT‐qPCR, to clarify associations of miRNA expression with clinicopathologic data and to evaluate the potential of miRNAs as diagnostic and prognostic markers. Matched tumor and adjacent normal tissues were obtained from 76 radical prostatectomy specimens. Twenty‐four tissue pairs were analyzed using human miRNA microarrays for 470 human miRNAs. Differentially expressed miRNAs were validated by TaqMan RT‐qPCR using all 76 tissue pairs. The diagnostic potential of miRNAs was calculated by receiver operating characteristics analyses. The prognostic value was assessed in terms of biochemical recurrence using Kaplan–Meier and Cox regression analyses. Fifteen differentially expressed miRNAs were identified with concordant fold‐changes by microarray and RT‐qPCR analyses. Ten microRNAs (hsa‐miR‐16, hsa‐miR‐31, hsa‐miR‐125b, hsa‐miR‐145, hsa‐miR‐149, hsa‐miR‐181b, hsa‐miR‐184, hsa‐miR‐205, hsa‐miR‐221, hsa‐miR‐222) were downregulated and 5 miRNAs (hsa‐miR‐96, hsa‐miR‐182, hsa‐miR‐182*, hsa‐miR‐183, hsa‐375) were upregulated. Expression of 5 miRNAs correlated with Gleason score or pathological tumor stage. Already 2 microRNAs classified up to 84% of malignant and nonmalignant samples correctly. Expression of hsa‐miR‐96 was associated with cancer recurrence after radical prostatectomy and that prognostic information was confirmed by an independent tumor sample set from 79 patients. That was shown with hsa‐miR‐96 and the Gleason score as final variables in the Cox models build in the 2 patient sets investigated. Thus, differential miRNAs in prostate cancer are useful diagnostic and prognostic indicators. This study provides a solid basis for further functional analyses of miRNAs in prostate cancer.

Gene expression studies in prostate cancer tissue: which reference gene should be selected for normalization?

Using quantitative reverse transcription–polymerase chain reaction (RT-PCR), reference genes are utilized as endogenous controls for relative quantification of target genes in gene profiling studies. The suitability of housekeeping genes for that purpose in prostate cancer tissue has not been sufficiently investigated so far. The objective of this study was to select from a panel of 16 potential candidate reference genes the most stable genes for gene normalization. Expression of mRNA encoding ACTB, ALAS1, ALB, B2M, G6PD, GAPD, HMBS, HPRT1, K-ALPHA-1, POLR2A, PPIA, RPL13A, SDHA, TBP, UBC, and YWHAZ was examined in matched, microdissected malignant and nonmalignant tissue specimens obtained from 17 nontreated prostate carcinomas after radical prostatectomy by real-time RT-PCR. The genes studied displayed a wide expression range with cycle threshold values between 16 and 37. The expression was not different between samples from pT2 and pT3 tumors or between samples with Gleason scores <7 and ≥7 (P>0.05). ACTB, RPL13A, and HMBS showed significant differences (P<0.02 at least) in expressions between malignant and nonmalignant pairs. All other genes did not differ between the matched pairs, and the software programs geNorm and NormFinder were used to ascertain the most suitable reference genes from these candidates. HPRT1, ALAS1, and K-ALPHA-1 were calculated by both programs to be the most stable genes covering a broad range of expression. The expression of the target gene RECK normalized with HRPT1 alone and with the normalization factors generated by the combination of these three reference genes as well as with the unstable genes ACTB or RPL13A is given. That example shows the significance of using suitable reference genes to avoid erroneous normalizations in gene profiling studies for prostate cancer. The use of HPRT1 alone as a reference gene shown in our study was sufficient, but the normalization factors generated from two (HRPT1, ALAS1) or all three genes (HRPT1, ALAS1, K-ALPHA-1) should be considered for an improved reliability of normalization in gene profiling studies of prostate cancer.

MicroRNA profiling of clear cell renal cell cancer identifies a robust signature to define renal malignancy

MicroRNAs are short single‐stranded RNAs that are associated with gene regulation at the transcriptional and translational level. Changes in their expression were found in a variety of human cancers. Only few data are available on microRNAs in clear cell renal cell carcinoma (ccRCC). We performed genome‐wide expression profiling of microRNAs using microarray analysis and quantification of specific microRNAs by TaqMan real‐time RT‐PCR. Matched malignant and non‐malignant tissue samples from two independent sets of 12 and 72 ccRCC were profiled. The microarray‐based experiments identified 13 over‐expressed and 20 down‐regulated microRNAs in malignant samples. Expression in ccRCC tissue samples compared with matched non‐malignant samples measured by RT‐PCR was increased on average by 2.7‐ to 23‐fold for the hsa‐miR‐16, −452*, −224, −155 and −210, but decreased by 4.8‐ to 138‐fold for hsa‐miR‐200b, −363, −429, −200c, −514 and −141. No significant associations between these differentially expressed microRNAs and the clinico‐pathological factors tumour stage, tumour grade and survival rate were found. Nevertheless, malignant and non‐malignant tissue could clearly be differentiated by their microRNA profile. A combination of miR‐141 and miR‐155 resulted in a 97% overall correct classification of samples. The presented differential microRNA pattern provides a solid basis for further validation, including functional studies.

Robust MicroRNA Stability in Degraded RNA Preparations from Human Tissue and Cell Samples

BACKGROUND RNA integrity is the essential factor that determines the accuracy of mRNA transcript measurements obtained with quantitative real-time reverse-transcription PCR (RT-qPCR), but evidence is clearly lacking on whether this conclusion also applies to microRNAs (miRNAs). We evaluated this issue by comparative analysis of the dependence of miRNA and mRNA measurements on RNA integrity in renal and prostate samples, under both model and clinical conditions. METHODS Samples of total RNA isolated from human renal tissue and Caki-2 cells, as well as from prostate tissue and LNCaP cells, were incubated at 80 degrees C for 5-240 min. We subsequently determined the RNA integrity number (RIN) and used RT-qPCR to measure various miRNAs (miR-141, miR-155, miR-200c, and miR-210 in renal samples, and miR-96, miR-130b, miR-149, miR-205, and miR-222 in prostate samples). We similarly measured mRNAs encoded by CDH16 (cadherin 16, KSP-cadherin), PPIA [peptidylprolyl isomerase A (cycophilin A)], and TBP (TATA box binding protein) in renal samples, and HIF1A [hypoxia-inducible factor 1, alpha subunit (basic helix-loop-helix transcription factor)], HPRT1 (hypoxanthine phosphoribosyltransferase 1), and KLK3 (kallikrein-related peptidase 3; also known as PSA) in prostate samples. Additionally, we quantified selected miRNAs and mRNAs in samples of RNAs with different RIN values that we isolated from clinical samples. The effect of RIN on the miRNA and mRNA data was assessed by linear regression analysis and group comparison. RESULTS The heat-incubation experiments of cell line and tissue RNAs showed that RIN values had negligible or no effect on miRNA results, whereas all mRNAs gradually decreased with decreasing RIN values. These findings were corroborated by our findings with clinical samples. CONCLUSIONS Our results suggest the stability of miRNAs to be generally robust, which makes feasible accurate miRNA measurements with RT-qPCR, even in degraded RNA preparations for which reliable mRNA analyses are commonly inapplicable.

In search of suitable reference genes for gene expression studies of human renal cell carcinoma by real-time PCR

BackgroundHousekeeping genes are commonly used as endogenous reference genes for the relative quantification of target genes in gene expression studies. No conclusive systematic study comparing the suitability of different candidate reference genes in clear cell renal cell carcinoma has been published to date. To remedy this situation, 10 housekeeping genes for normalizing purposes of RT-PCR measurements already recommended in various studies were examined with regard to their usefulness as reference genes.ResultsThe expression of the potential reference genes was examined in matched malignant and non-malignant tissue specimens from 25 patients with clear cell renal cell carcinoma. Quality assessment of isolated RNA performed with a 2100 Agilent Bioanalyzer showed a mean RNA integrity number of 8.7 for all samples. The between-run variations related to the crossing points of PCR reactions of a control material ranged from 0.17% to 0.38%. The expression of all genes did not depend on age, sex, and tumour stage. Except the genes TATA box binding protein (TBP) and peptidylprolyl isomerase A (PPIA), all genes showed significant differences in expression between malignant and non-malignant pairs. The expression stability of the candidate reference genes was additionally controlled using the software programs geNorm and NormFinder. TBP and PPIA were validated as suitable reference genes by normalizing the target gene ADAM9 using these two most stably expressed genes in comparison with up- and down-regulated housekeeping genes of the panel.ConclusionOur study demonstrated the suitability of the two housekeeping genes PPIA and TBP as endogenous reference genes when comparing malignant tissue samples with adjacent normal tissue samples from clear cell renal cell carcinoma. Both genes are recommended as reference genes for relative gene quantification in gene profiling studies either as single gene or preferably in combination.

Changes in Concentration of DNA in Serum and Plasma during Storage of Blood Samples

Cell-free DNA in serum and plasma has been suggested to have diagnostic potential because associations between DNA concentrations and several disorders have been described (1). The concentration of cell-free DNA circulating in plasma and serum has been analyzed in several studies and used as an interchangeable index of the quantity of circulating DNA in blood (2)(3)(4). However, it is known that the DNA concentration in serum is ∼3- to 24-fold higher than in plasma (3)(4)(5)(6). Recently published articles in this journal showed that various preanalytical factors of blood sampling and processing can affect the DNA concentration in plasma (5)(7)(8), but these findings do not explain the difference between plasma and serum concentrations of DNA mentioned above. Comparative investigations of the preanalytical conditions influencing the DNA concentration in serum and plasma are lacking. In addition, reference intervals for the concentration of cell-free DNA in serum were established without considering these factors (9). Thus, to complement the data of Lui et al. (5), we analyzed the influence of time delay in blood processing for plasma and serum at room temperature and at 4 °C. Venous blood samples from 10 healthy volunteers (5 females and 5 males; mean age, 42 years) were simultaneously collected in …

Identification and Validation of Suitable Endogenous Reference Genes for Gene Expression Studies of Human Bladder Cancer

PURPOSE Housekeeping genes as endogenous references are generally used for the relative quantification of target genes in gene profiling studies. To date that issue has not been sufficiently investigated in bladder cancer. From a panel of 9 potential candidates we selected the most stable housekeeping genes for gene normalization in bladder cancer tissue. MATERIALS AND METHODS Expression profiles of the 9 genes ACTB, ALAS1, G6PD, GAPD, HMBS, HPRT1, K-ALPHA-1, SDHA and TBP were established in matched malignant and nonmalignant tissue specimens from 14 patients with bladder cancer. Quality assessment of isolated RNA was performed with a 2100 Bioanalyzer (Agilent Technologies, Palo Alto, California) and real-time reverse transcriptase-polymerase chain reaction was performed with LightCycler. The software geNorm and NormFinder (Aarhus University, Aarhus, Denmark) were used to identify the most suitable reference genes. RESULTS RNA was isolated with high purity and integrity. Candidate reference genes showed a broad range of between 20 and 34 polymerase chain reaction cycles. Expression did not depend on patient sex or tumor stage. GAPD, G6PD and HMBS showed significant differences in expression between malignant and nonmalignant pairs (at least p <0.04). Expression of the remaining genes did not differ between the matched pairs. SDHA and TBP were the most stably expressed genes, covering higher and lower expression levels. CONCLUSIONS For normalization purposes in gene profiling studies of bladder cancer the genes SDH and TBP are recommended as single reference genes depending on the expression level of the target gene or more favorably in combination.

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.

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.

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.