MiRNA-141 and miRNA-200b are closely related to invasive ability and considered as decision-making biomarkers for the extent of PLND during cystectomy

This study was approved by the Ethics Committee of Central South University, Changsha city, Hunan province, China. Informed consent was obtained from all of the patients. The methods were carried out in accordance with the approved guidelines. Specimens of BC and corresponding adjacent tissues were collected from 30 patients with muscle-invasive BC who underwent cystectomy at Xiangya hospital, Central South University.

To investigate the differential expression of five members of the miRNA-200 family in BC tissues and adjacent tissues, we analyzed 60 fresh bladder tissues (30 BC specimens and 30 corresponding adjacent tissues) using qRT-PCR. For RNA extraction, bladder tissues with a diameter of about 3–4 mm were used for RNA extraction with a mirVana™ miRNA Isolation Kit, following the instructions of the manufacturer (Applied Biosystems, USA). For detection of miRNAs, qRT-PCR was performed using TaqMan miRNA assays (Applied Biosystems, USA) with specific commercial primer sets and probes. All reagents and protocols were obtained from Applied Biosystems and detection was performed using U6 as an internal control. Comparative quantification was performed on the basis of a 2^−ΔΔCt calculation method [18]. The miRNA specific qRT-PCR was done in triplicate and repeated three times.

The human bladder carcinoma cell lines CRL 1749, J82, T24, HT 1376 and HTB 9 were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum and penicillin/streptomycin.

To explore the function of endogenous miRNA-141 and miRNA-200b, we generated constructs of pri- or anti-miRNA, aiming either to increase or to inhibit the function of the molecules. A miRNA sponge was initially developed by Ebert and colleagues to inhibit miRNA function [8]. The pLVX-IRES-ZsGreen1 expression vector was purchased from Clontech Laboratories, Inc (USA).

Lentiviral vectors of pLVX-pri-miRNA-141 or pLVX-pri-miRNA-200b which expressed miRNA-141 or miRNA-200b were constructed (Yinrunbio, Changsha, China). Sponge plasmids, pLVX-miRNA141-sponge and pLVX-miRNA200b-sponge, were constructed as described by Ebert et al. [8].

A pre-mixed Lentiviral Packaging System (Biosettia, SD, USA) was used for viral packaging. Briefly, lentivirus was produced by transfection of 293 T cells at 5 × 10⁶ cells/10 cm plate using Lipofectamine 2000. Supernatants were collected 48 h after transfection and filtered; the viral titers were determined by fluorescence-activated cell sorting (FACS) at 48 h post-transduction. The BC cells were infected with lentivirus in the presence of 8 μg/ml polybrene (Sigma-Aldrich, USA). The green fluorescence protein (ZsGreen1), which was co-expressed in lentivirus-infected cells, served as a selection marker to indicate the successfully infected cancer cells.

The invasive capacity of the cells was determined using BD BioCoat Matrigel invasion chambers (8-μm pores) (BD Biosciences, USA). The cells were seeded on the top chamber, incubated at 37°C, and allowed to invade and migrate through the Matrigel and the membrane pores in the inserts. After 48 h, the cells on the surface of the membrane were wiped off. The cells on the underside of the membrane were fixed and stained. The invasive cells were counted under a microscope at 100× magnification.

Total RNA from cell samples was extracted using an RNeasy Mini Kit (Qiagen, USA). Complementary DNA (cDNA) was synthesized using the High-Capacity cDNA archive kit (Applied Biosystems, USA), according to the manufacturer’s protocol. The cDNA was synthesized from 2 μg total RNA on a PTC-200 Peltier Thermal Cycler DNA Engine (MJ Research Inc., USA). The DNA Engine Thermal Cycler with Chromo 4™ real-time detector system and Opticon Monitor software (Bio-Rad Laboratories, USA) were used for real-time PCR analysis. Cycle threshold (Ct) values were normalized to the housekeeper GAPDH gene.

The specific primers used were as follows: GAPDH (forward: 5′-ACCACAGTCCATGCCAT CAC-3′; reverse: 5′-TCCACCACCCTGTTGCTGTA-3′); N-cadherin (forward: 5′-AACCCTTATTTT GCCCCCAAT-3′; reverse: 5′-TCAACATGGTACCGGCATGA-3′); E-cadherin (forward: 5′-CGGGA ATGCAGTTGAGGATC-3′; reverse: 5′-AGGATGGTGTAAGCGATGGC-3′); vimentin (forward: 5′-GACCTCTACGAGGAGGAGAT-3′; reverse: 5′-TTGTCAACATCCTGTCTGAA-3′).

Cultured cells were directly lysed for 30 minutes on ice with lysis buffer [50 mmol/L Tris–HCl (pH 7.4), 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L PMSF, 1 μg/mL aprotinin, 1 μg/mL leupeptin, 1 μg/mL pepstatin, 1 mmol/L Na₃VO₄ and 1 mmol/L NaF]. After centrifugation at 13,000 g for 15 min, protein concentrations were measured using Bradford’s reagent (Bio-Rad laboratories, USA), and the protein was denatured by boiling for 10 min. Protein (25 μg) was loaded onto sodium dodecylsulfate–polyacrylamide gels for electrophoresis and then transferred onto nitrocellulose membranes. After blocking with 5% milk in TBST (137 mmol/L NaCl, 25 mmol/L Tris, and 1 mmol/L disodium ethylenediaminotetraacetate containing 0.1% Tween-20), the membranes were incubated with anti-E-cadherin, anti-vimentin (Cell Signaling Technology, USA), anti-matrix metalloproteinase (MMP)-2 and MMP-9 (Chemicon, USA), anti-N-cadherin (Santa Cruz Biotechnology, USA), and anti-GAPDH (Santa Cruz Biotechnology, USA) at 4°C overnight. After washing with TBST three times (10 min each), the membranes were incubated with their corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies at room temperature for 1 h. After washing with TBST three times (10 min each), bound antibodies were visualized using enhanced chemiluminescent substrates (Amersham Bioscience, USA).

Gelatine zymography was used to evaluate the levels of expression of MMPs in conditioned media from cultured cells. To generate supernatants for zymography, cells were seeded in six-well plates with complete medium. On the following day, the medium was replaced by starving medium containing 0.1% BSA. After incubation for 24 h, supernatants were collected, centrifuged and analyzed by zymography. The conditioned medium was electrophoresed in a polyacrylamide gel containing gelatin at a concentration of 1 mg/mL. The gel was washed at room temperature for 2 h with 2.5% Triton X-100 and then at 37°C overnight in a buffer containing 10 mmol CaCl2, 150 mm NaCl and 50 mmol Tris–HCl (pH 7.5). For visualization of gelatinolytic activity, the gels were stained with Coomassie blue and photographed on a light box. Proteolysis was detected as a white zone in a dark blue field.

Urine samples from 78 patients with BC were obtained from our center between January 2010 and March 2013. Agreement of collecting their urine and written informed consent have been obtained from all included patients. All patients underwent laparoscopic cystectomy accompanied by super-extended PLND as described in a previous report [19]. Their average age was 57 + 12.7 years. Sixty-four (82.1%) of the patients were male and 14 (17.9%) were female. Radiological tests, including chest X-ray and CT, were routinely done according to EAU guidelines [20]. Preoperative CT staging of bladder tumors was evaluated using a standardized method [19]. Histopathological examination of bladder cancer specimens and lymphatic tissues obtained during lymph node dissection was performed by experienced genitourinary pathologists. The region of lymph node metastasis (LN+) was divided into level I, level II and level III, as introduced by Leissner et al. [8].

Total urine samples (100–150 mL) were collected before cystectomy. The urine was stored at 4°C for up to 4 h and then centrifuged. The pellet was re-suspended in 1 ml of TRIzol reagent and frozen at −80 C in liquid nitrogen for further use. RNAs were extracted from the urinary cell pellets using a mirVana™ miRNA Isolation Kit, following the instructions of the manufacturer (Applied Biosystems, USA). The expression of miRNA-200b and miRNA-141 was detected using qRT-PCR as described above.

A two-tailed chi-square test was used to determine the statistical significance of differences between proportions. The Mann–Whitney U test or Wilcoxon signed-rank test was used for continuous variables. The value of each biomarker in deciding the extent of PLND was evaluated by calculating the ROC AUC. The ROC AUC of each model was compared using the DeLong test [21]. P values less than 0.05 were counted as significant in all tests. The statistical analysis was performed using SPSS for Windows v.13.0 and MedCalc statistical software 11.5.0.