Cell-free urinary microRNA-99a and microRNA-125b are diagnostic markers for the non-invasive screening of bladder cancer

Ding-Zuan Zhang, Kin-Mang Lau, Eddie S Y Chan, Gang Wang, Cheuk-Chun Szeto, Kenneth Wong, Richard K W Choy, Chi-Fai Ng, Ding-Zuan Zhang, Kin-Mang Lau, Eddie S Y Chan, Gang Wang, Cheuk-Chun Szeto, Kenneth Wong, Richard K W Choy, Chi-Fai Ng

Abstract

Background: Evidence implicated the diagnostic significance of microRNAs in whole urine/urine sediments in urothelial carcinoma of the bladder (UCB). However, the contaminated blood cells in patients with haematouria significantly altered the expression profiles of urinary microRNA, influencing the test accuracy.

Methods: MicroRNA profiles of the urine supernatants of UCB patients and controls without any malignancy and profiles of malignant and corresponding normal mucosa tissues from the patients were determined by microRNA microarray and compared to identify differentially expressed microRNAs. The differential expression was verified in the tissues of an independent patient cohort by RT-qPCR. The diagnostic significance of selected microRNAs as biomarkers in the urine supernatant was investigated in the expanded cohorts.

Results: MicroRNA-99a and microRNA-125b were down-regulated in the urine supernatants of UCB patients. The degree of down-regulation was associated with the tumor grade. A diagnostic model was developed using a combined index of the levels of microRNA-99a and microRNA-125b in the urine supernatant with a sensitivity of 86.7%, a specificity of 81.1% and a positive predicted value (PPV) of 91.8%. Discriminating between high- and low-grade UCB, the model using the level of microRNA-125b alone exhibited a sensitivity of 81.4%, a specificity of 87.0% and a PPV of 93.4%.

Conclusions: The results revealed a unique microRNA expression signature in the urine supernatants of UCB patients for the development of molecular diagnostic tests. An effective cell-free urinary microRNA-based model was developed using a combined index of the levels of microRNA-99a and microRNA-125b to detect UCB with good discriminating power, high sensitivity and high specificity.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Results of microRNA microarray analysis.
Figure 1. Results of microRNA microarray analysis.
(A) Heat map of the levels of 10 selected microRNAs in nine urine supernatant samples and four pairs of tissue samples. The total RNA was extracted and subjected to microRNA microarray analysis using an Agilent Human microRNA Microarray Chip. The signal intensity of each spot was median-normalized and further normalized by linear regression. The normalized signal intensities for each microRNA in the urine supernatant and tissue samples of UCB patients and controls are presented. (B) Scatter plots of the normalized signal sensitivities of the 10 selected microRNAs. A Mann-Whitney U test was conducted to compare the levels of microRNAs in the urine supernatant of UCB patients (TU) (n = 6) and controls (NU) (n = 3), and a paired Mann-Whitney U test was conducted to compare the tumors (TT) (n = 4) and their corresponding normal mucosa tissues (NT) (n = 4) in the UCB patients. A significance level of 0.01 was used for all of the comparisons. Asterisks (*) indicate a statistically significant difference (p

Figure 2. Validation study of the down-regulated…

Figure 2. Validation study of the down-regulated expression of 10 selected microRNAs in bladder cancer…

Figure 2. Validation study of the down-regulated expression of 10 selected microRNAs in bladder cancer tissues by RT-qPCR.
The total RNA was extracted from 18 pairs of bladder cancer tissues and their corresponding normal adjacent mucosa tissues. The levels of these microRNAs were quantified by RT-qPCR in duplicate. The relative expression levels (2-ΔCq) are presented. A paired Mann-Whitney U test was conducted to compare the tumors and their corresponding normal mucosa tissues in UCB patients. Columns, Means; Bars, S.D.; n = 18. ** denotes p

Figure 3. Expression of microRNAs in the…

Figure 3. Expression of microRNAs in the urine supernatant of UCB patients and normal controls.

Figure 3. Expression of microRNAs in the urine supernatant of UCB patients and normal controls.
The total RNA was extracted from the urine supernatants of UCB patients with low-grade (n = 15) (shaded bar) or high-grade cancers (n = 35) (black bar) and normal controls (n = 21) (white bar). The levels of six microRNAs in the samples were quantified by RT-qPCR in duplicate. (A) The relative levels (2-ΔCq) of microRNA-133a, microRNA99a, microRNA-1, microRNA-143, microRNA-133b and microRNA-125b in the urine supernatant samples are presented. A Mann-Whitney U test was conducted to compare the UCB patients and normal controls. Columns, Means; Bars, S.D.; ** denotes phsa-miR-99a and hsa-miR-125b-2). Human Genome Reference Consortium GRCh37 Patch Release 12 assembly was used. Hsa-miR-99a is located at chromosome 21:17,909,409–17,913,489, and hsa-miR-125b-2 is located at chromosome 21:17,960,557–17,964,645.

Figure 4. Restored expression of microRNA-99a and…

Figure 4. Restored expression of microRNA-99a and microRNA-125b in the urine supernatant of UCB patients…

Figure 4. Restored expression of microRNA-99a and microRNA-125b in the urine supernatant of UCB patients after transurethial resection.
Urine samples were collected from 20 UCB patients before (pre-operative) and after (post-operative) transurethial resection. The total RNA was extracted from the urine supernatants and subjected to RT-qPCR to quantify the levels of microRNA-99a (left) and microRNA-125b (right). A Wilcoxon signed-rank two-related-samples test was conducted to compare the levels of microRNA-99a and microRNA-125b between the pre- and post-operative samples. * denotes p
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References
    1. Timberg G, Rahu M, Gornoi K, Aareleid T, Baburin A (1997) Bladder cancer in Estonia, 1968–1992: incidence, mortality, prevalence and survival. Scand J Urol Nephrol 31: 337–342. - PubMed
    1. Parker J, Spiess PE (2011) Current and emerging bladder cancer urinary biomarkers. Sci World J 11: 1103–1112. - PMC - PubMed
    1. Fabbri M, Garzon R, Cimmino A, Liu Z, Zanesi N, et al. (2006) MicroRNA expression and function in cancer. Trends Mol Med 12: 580–587. - PubMed
    1. Iorio MV, Ferracin M, Liu CG, Veronese A, Spizzo R, et al. (2005) MicroRNA gene expression deregulation in human breast cancer. Cancer Res 65: 7065–7070. - PubMed
    1. Yanaihara N, Caplen N, Bowman E, Seike M, Kumamoto K, et al. (2006) Unique microRNA molecular profiles in lung cancer diagnosis and prognosis. Cancer Cell 9: 189–198. - PubMed
Show all 35 references
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This study was supported by the Direct Grant for Research (2009.1.096), The Chinese University of Hong Kong. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Figure 2. Validation study of the down-regulated…
Figure 2. Validation study of the down-regulated expression of 10 selected microRNAs in bladder cancer tissues by RT-qPCR.
The total RNA was extracted from 18 pairs of bladder cancer tissues and their corresponding normal adjacent mucosa tissues. The levels of these microRNAs were quantified by RT-qPCR in duplicate. The relative expression levels (2-ΔCq) are presented. A paired Mann-Whitney U test was conducted to compare the tumors and their corresponding normal mucosa tissues in UCB patients. Columns, Means; Bars, S.D.; n = 18. ** denotes p

Figure 3. Expression of microRNAs in the…

Figure 3. Expression of microRNAs in the urine supernatant of UCB patients and normal controls.

Figure 3. Expression of microRNAs in the urine supernatant of UCB patients and normal controls.
The total RNA was extracted from the urine supernatants of UCB patients with low-grade (n = 15) (shaded bar) or high-grade cancers (n = 35) (black bar) and normal controls (n = 21) (white bar). The levels of six microRNAs in the samples were quantified by RT-qPCR in duplicate. (A) The relative levels (2-ΔCq) of microRNA-133a, microRNA99a, microRNA-1, microRNA-143, microRNA-133b and microRNA-125b in the urine supernatant samples are presented. A Mann-Whitney U test was conducted to compare the UCB patients and normal controls. Columns, Means; Bars, S.D.; ** denotes phsa-miR-99a and hsa-miR-125b-2). Human Genome Reference Consortium GRCh37 Patch Release 12 assembly was used. Hsa-miR-99a is located at chromosome 21:17,909,409–17,913,489, and hsa-miR-125b-2 is located at chromosome 21:17,960,557–17,964,645.

Figure 4. Restored expression of microRNA-99a and…

Figure 4. Restored expression of microRNA-99a and microRNA-125b in the urine supernatant of UCB patients…

Figure 4. Restored expression of microRNA-99a and microRNA-125b in the urine supernatant of UCB patients after transurethial resection.
Urine samples were collected from 20 UCB patients before (pre-operative) and after (post-operative) transurethial resection. The total RNA was extracted from the urine supernatants and subjected to RT-qPCR to quantify the levels of microRNA-99a (left) and microRNA-125b (right). A Wilcoxon signed-rank two-related-samples test was conducted to compare the levels of microRNA-99a and microRNA-125b between the pre- and post-operative samples. * denotes p
Similar articles
Cited by
References
    1. Timberg G, Rahu M, Gornoi K, Aareleid T, Baburin A (1997) Bladder cancer in Estonia, 1968–1992: incidence, mortality, prevalence and survival. Scand J Urol Nephrol 31: 337–342. - PubMed
    1. Parker J, Spiess PE (2011) Current and emerging bladder cancer urinary biomarkers. Sci World J 11: 1103–1112. - PMC - PubMed
    1. Fabbri M, Garzon R, Cimmino A, Liu Z, Zanesi N, et al. (2006) MicroRNA expression and function in cancer. Trends Mol Med 12: 580–587. - PubMed
    1. Iorio MV, Ferracin M, Liu CG, Veronese A, Spizzo R, et al. (2005) MicroRNA gene expression deregulation in human breast cancer. Cancer Res 65: 7065–7070. - PubMed
    1. Yanaihara N, Caplen N, Bowman E, Seike M, Kumamoto K, et al. (2006) Unique microRNA molecular profiles in lung cancer diagnosis and prognosis. Cancer Cell 9: 189–198. - PubMed
Show all 35 references
Publication types
MeSH terms
Related information
Grant support
This study was supported by the Direct Grant for Research (2009.1.096), The Chinese University of Hong Kong. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
[x]
Cite
Copy Download .nbib
Format: AMA APA MLA NLM
Figure 3. Expression of microRNAs in the…
Figure 3. Expression of microRNAs in the urine supernatant of UCB patients and normal controls.
The total RNA was extracted from the urine supernatants of UCB patients with low-grade (n = 15) (shaded bar) or high-grade cancers (n = 35) (black bar) and normal controls (n = 21) (white bar). The levels of six microRNAs in the samples were quantified by RT-qPCR in duplicate. (A) The relative levels (2-ΔCq) of microRNA-133a, microRNA99a, microRNA-1, microRNA-143, microRNA-133b and microRNA-125b in the urine supernatant samples are presented. A Mann-Whitney U test was conducted to compare the UCB patients and normal controls. Columns, Means; Bars, S.D.; ** denotes phsa-miR-99a and hsa-miR-125b-2). Human Genome Reference Consortium GRCh37 Patch Release 12 assembly was used. Hsa-miR-99a is located at chromosome 21:17,909,409–17,913,489, and hsa-miR-125b-2 is located at chromosome 21:17,960,557–17,964,645.
Figure 4. Restored expression of microRNA-99a and…
Figure 4. Restored expression of microRNA-99a and microRNA-125b in the urine supernatant of UCB patients after transurethial resection.
Urine samples were collected from 20 UCB patients before (pre-operative) and after (post-operative) transurethial resection. The total RNA was extracted from the urine supernatants and subjected to RT-qPCR to quantify the levels of microRNA-99a (left) and microRNA-125b (right). A Wilcoxon signed-rank two-related-samples test was conducted to compare the levels of microRNA-99a and microRNA-125b between the pre- and post-operative samples. * denotes p

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