The serum and urine samples were obtained from 17 patients (4 women and 13 men) admitted to the urology department. The age range was 38–84 years with an average age of 65.7. The majority of the patients were male (76%). Twelve patients (71%) in the group were diagnosed with non-invasive bladder cancer, and the remaining 5 patients (29%) had invasive bladder cancer in stage pT2. None of the patients were diagnosed with CIS (carcinoma in situ). Tumor histological grade was determined according to the World Health Organization/International Society of Urological Pathology criteria of 2004, and tumor stage was assessed in accordance with the 2002 UICC TNM classification [24, 25]. The control group consisted of 3 samples isolated from oncologically healthy patients admitted to the urology ward. They underwent cystoscopy excluding tumor changes in the bladder.
Characteristics of tested population is shown in Table 1 (Table 1).
Table 1
Characteristic of tested population
| | Results |
Gender | Male | 13 (76%) |
| Female | 4 (24%) |
Tumor grade | Low-grade | 10 (59%) |
| High-grade | 7 (41%) |
Tumor stage | pTa | 10 (59%) |
| pT1 | 2 (12%) |
| pT2 | 5 (29%) |
Smoking history | | 12 (70%) |
Occupational exposure | | 5 (29%) |
Age [years] | minimum | 38 |
| maximum | 84 |
| average | 65.7 |
The inclusion criteria for the study of the tested group comprised age over 18 years old, newly-diagnosed bladder cancer and the ability to give informed consent. The exclusion criterion was the history of bladder cancer in the past or recurrent bladder cancer and history of any other neoplasm. Inclusion criteria for control group were absence of current bladder tumor and no history of bladder cancer nor any other neoplasm.
The permission to conduct the study was granted by the Bioethics Advisory Commission at Lodz Medical University, No. RNN/231/17/KE, and all the patients signed consent forms. The study was supported by Medical University Grant No. 502-03/5-138-02/502-54-146.
Urine and blood samples were collected before TURBT, and were stored for less than four hours, at + 4 °C, before miRNA isolation. Urine and blood samples were centrifuged for 15 minutes at 3000 rpm and the resulting urine supernatant or plasma were stored at -20 °C.
We applied the isolation protocol of TriPure Isolation Reagent, Roche, Cat No. 11 667 165001, Version 07 which was used to isolate miRNAs. The amount and quality of isolated RNA was measured spectrophotometrically.[26] In addition, the quality of the obtained isolate was checked electrophoretically on a 2% agarose gel, which allowed for assessment of the degree of RNA degradation. Qualitative and quantitative assessment of DNA and RNA was made on the basis of spectrophotometric measurement of absorbance at 260 nm, 280 nm, and 230 nm light length. The measurement was performed using the Implen NanoPhotometer NP80 device.
For real time PCR (Reverse Transcription Reaction), we used the protocol for High- Capacity cDNA Reverse Transcription Kits, Applied Biosystems, Cat No. 4368813, Lot No. 1201113. Random pairs of primers were used in the kit, ensuring synthesis efficiency for all RNA molecules present in the sample, including mRNA and rRNA. To assess the expression of individual microRNAs in the serum and urine supernatant, real-time PCR was performed with primers specific for miRNAs 106b-3p, 130b-3, 145-3p, and 199a-5p using the miRCURY LNA Universal RT microRNA PCR set (Exiqon. Cat No. 203351, T100 Thermal Cycler, Bio-Rad), following the manufacturer’s protocol. Reactions were carried out in special 96-well plates on a BIO-RAD CFX 96 Real-Time System device. Each reaction was performed twice, and the arithmetic average of these two measurements was taken for further analysis.
Expression levels for the 106b-3p, 130b-3, 145-3p, 199a-5p microRNA were assessed using a comparative method, also known as the double delta (ΔΔCt) method which is used to determine the relative difference in the expression level between tested samples and the reference. The first stage consists of the analysis of the marked Ct (the cycle at which the fluorescence level reaches a certain amount) in the amplification reaction of the examined miRNAs and control miRNA for both the tested and the control groups.
(∆Ct) (tested group) = Ct miRNA target – Ct miRNA reference
(∆Ct) (control group) = Ct miRNA target – Ct miRNA reference
Next, ∆∆Ct was calculated for each sample:
∆∆Ct = ∆Ct (tested samples) – ∆Ct (control group)
The calculation of the relative expression level (R) of the tested marker in the tested sample against the control sample was made using a formula: R = 2 −∆∆Ct
The ΔΔCt method assumes a uniform PCR amplification efficiency of 100% across all samples. In our study, the eciency was between 98.9% and 100%. The value of R = 1 means that the level of microRNA expression in the test is the same as in the calibration sample. A value of R > 1 indicates that the level of expression in the test sample is higher than the calibration sample. A value R < 1 means lower expression level in the test sample than in the calibration sample. [27, 28]
The program PQStat version 1.6.8.304. was used to make statistical calculations. The analysis was performed with the Mann-Whitney U test. The value p < 0.05 was accepted as the threshold of statistical difference.