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Detection of let-7 miRNAs in urine supernatant as potential diagnostic approach in non-metastatic clear-cell renal cell carcinoma

Introduction Urinary microRNAs (miRNAs) are emerging as a clinically useful tool for early and non-invasive detection of various types of cancer. The aim of this study was to evaluate whether let-7 family miRNAs differ in their urinary concentrations between renal cell carcinoma (RCC) cases and healthy controls. Materials and methods In the case-control study, 69 non-metastatic clear-cell RCC patients and 36 gender/age-matched healthy controls were prospectively enrolled. Total RNA was purified from cell-free supernatant of the 105 first morning urine specimens. Let-7 family miRNAs were determined in cell-free supernatant using quantitative miRNA real-time reverse-transcription PCR and absolute quantification approach. Results Concentrations of all let-7 miRNAs (let-7a, let-7b, let-7c, let-7d, let-7e and let-7g) were significantly higher in urine samples obtained from RCC patients compared to healthy controls (P < 0.001; P < 0.001; P = 0.005; P = 0.006; P = 0.015 and P = 0.002, respectively). Subsequent ROC analysis has shown that let-7a concentration possesses good ability to differentiate between cases and controls with area under curve being 0.8307 (sensitivity 71%, specificity 81%). Conclusions We have shown that let-7 miRNAs are abundant in the urine samples of patients with clear-cell RCC, and out of six let-7 family members, let-7a outperforms the others and presents promising non-invasive biomarker for the detection of RCC.


Introduction
Renal cell carcinoma (RCC) accounts for 2-3% of all malignant tumours. There are several histological subtypes of RCC, with clear-cell (or conventional) histological type being the most frequent, presenting 70% -80% of RCC cases (1). Despite a steady decrease in mortality rates, RCC remains one of the most lethal urological malignancies, with 5-year relative survival 72% (1). For advanced and metastatic RCCs (32% of all diagnosed cases), 5-year relative survival descends to 66% and 12%, respectively. Biomarkers for early detection of RCC are therefore necessary as there is no reliable diagnostic modality other than radiological imaging.
MicroRNAs (miRNAs) are short noncoding RNAs that regulate gene expression at the posttranscriptional level. They are involved in the number of critical biological processes including carcinogenesis. Besides tumour tissues, they are also present in different body fluids (e.g. serum, plasma, urine) with a high degree of stability indicating their extensive biomarker potential (2). Although variety of circulating miRNAs has been proposed as bio- Fedorko M. et al. Urinary let-7 miRNAs in renal cell carcinoma markers of RCC, urinary miRNAs have been mostly studied in bladder and prostate cancer (3).
The miRNA let-7 family is widely accepted as a tumour suppressor miRNA with important role in the regulation of cell cycle, cell differentiation and apoptosis (4). Downregulation of the members of let-7 family has been observed in various types of tumour tissue including RCC (5). Less frequent, upregulation of certain let-7 family members has also been observed, suggesting that let-7 does not play a tumour suppressor function under all circumstances and in all tissues (6). Higher levels of let-7 miRNAs in urine has been reported in bladder cancer (7). There are no data about urinary let-7 miRNAs in RCC, but increased urinary levels of some of the let-7 family members were found in patients with autosomal dominant polycystic kidney disease suggesting their abundance in urine and therefore also potential utility in other renal diseases such as cancer (8).
Based on that, we hypothesize, that let-7 family miRNAs differ in their concentrations in urine samples of RCC patients and heathy individuals, and could be potentially useful as diagnostic biomarkers of RCC. To this end, the aim of our study was to determine concentration of let-7 miRNAs in cellfree supernatant in group of prospectively enrolled patients with non-metastatic clear cell RCC and group of healthy controls, statistically evaluate the differences in concentrations between the groups and their ability to distinguish between RCC cases and healthy controls.

Urine sampling and RNA isolation
The first morning urine samples were collected in 15 mL tubes (Sarstedt AG & Co., Numbrecht, Germany) with EDTA used for nucleic acid preservation and kept at 4 o C till further processing. As the next step, urine samples were centrifuged at 4 °C at 2000g for 15 minutes, and the cell-free supernatant was then collected and stored at − 80 °C until analysis. Before RNA isolation another centrifugation of urine sample was performed at 4 °C at 12,000g for 15 minutes. Total RNA from 1 mL of cell-free supernatant was isolated using manual column-based method, Urine microRNA Purification Kit (Norgen Biotek, Thorold, Ontario, Canada) according to the manufacturer's instructions. RNA concentration and purity was evaluated using Na- We also included inter-plate calibrator on each plate for each assay enabling us to correct for inter-plate variability. Quantitatively all measurements were standardized by use of the same amount of total RNA (10 ng) entering the reverse transcription and PCR reaction. Ct values of biological samples were converted to absolute concentration of miRNAs in the cell-free supernatant of the urine (fmol/L) based on relevant calibration curve equation (Figure 1) based on the recently described approach (9).

Statistical analysis
Statistical analysis was performed with GraphPad Prism version 6.00 for Windows (GraphPad Software, La Jolla California USA, www.graphpad.com).
To compare urinary concentrations of miRNAs between RCC cases and healthy controls non-parametric Mann-Whitney U test was used since our experimental data do not follow a normal distribution. A P-value < 0.01 was considered statistically significant. The ROC analysis was performed to evaluate the ability of studied miRNAs to distinguish between urine of RCC patients and healthy controls.
Complementary DNA was synthesized from 10 ng of total RNA in 15-µL reverse transcription (RT) reaction using microRNA-specific stem-loop RT

Results
We successfully purified RNA from urine samples of 105 subjects enrolled into our study. The con-centration of RNA ranged from 5.1 to 17.3 with a median of 6.7 ng/µL and the purity (A260/A280) ranged from 1.3 to 2.0 with a median of 1.5.

Fedorko M. et al. Urinary let-7 miRNAs in renal cell carcinoma
The concentrations of all 6 miRNAs (let-7a, let-7b, let-7c, let-7d, let-7e, and let-7g) were significantly higher in urine samples obtained from RCC cases compared to healthy controls (P < 0.001; P < 0.001; P = 0.005; P = 0.006; P = 0.015 and P = 0.002, respectively; Table 2). Subsequent ROC analysis was performed to evaluate ability of urinary miRNAs to distinguish between RCC cases and controls. ROC curves indicated that urine concentration of let-7a possess satisfactory ability to differentiate between patients and controls with the AUC being 0.8307 (Figure 2A,B). The remaining let-7 miRNAs showed inferior analytical performance (AUC < 0.75; summarized in Table 2). We further evaluated analytical performance of combination of all let-7 miRNAs with AUC being 0.83.

Discussion
Noninvasive biomarker of RCC in urine presents a significant unmet medical need of urologic oncology. To prove our hypothesis that let-7 miRNAs concentrations in urine differ between RCC cases and healthy controls, we used the case-control design performed prospectively enabling us to control pre-analytical conditions, sample handling and processing. Common approaches to miRNA clinical testing include small RNA sequencing, qRT-PCR, miRNA microarray, multiplexed miRNA detection with color-coded probe pairs, and miRNA in situ hybridization. We decided to use qRT-PCR, since our approach is targeted and this method has several advantages in comparison to others:    high dynamic range, high sensitivity and specificity, small requests on RNA input, it is widely used in clinical diagnostics and comparatively inexpensive.
In our study, we confirmed our hypothesis, and found that urinary concentrations of let-7 miRNAs in RCC patients are significantly higher compared to healthy controls. Let-7a concentrations enabled to discriminate urine of the RCC patients and controls with a sensitivity of 71% and specificity of 81%, suggesting its diagnostic value for detection of RCC. We further evaluated analytical performance of combination of all let-7 miRNAs and there was no notable increase in AUC values observed in comparison to let-7a used as the only biomarker. Our study has several limitations, which should be discussed. The main limitation is the small group of RCC cases and controls and absence of the independent validation set. To this end, our study is a pilot study showing diagnostic potential of urinary let-7a concentrations in detection of RCC, but further independent studies are needed to confirm our results. Another limitation is absolute quantification approach, which we used for determination of studied urinary miRNAs disabling to eliminate methodical inaccuracies, which could occur in processing of every sample and, finally, could bias comparisons of different groups of samples. There were some transcripts used for normalization of urinary miRNAs (e.g. RNU6B or RNU48) (3), but to our knowledge there is no consensual reference gene. Therefore, we decided to use absolute quantification and to overcome this potential technological variability or bias, we implemented standardized protocols for urine samples collection, handling and storage.
In conclusion, we showed that let-7 miRNA family members are abundant in the urine cell-free supernatant of patients with clear-cell RCC, and confirmed our hypothesis, that let-7 miRNAs have different concentrations in the urine of RCC cases and healthy controls. Out of six let-7 members analysed, let-7a outperforms the others and may be considered as a promising noninvasive biomarker for the detection of clear-cell RCC.