ABSTRACT
BACKGROUND: DNA methylation in cancer is considered a diagnostic and predictive biomarker. We investigated the usefulness of the methylation status of CALN1 as a biomarker for bladder cancer using methylation-sensitive restriction enzyme (MSRE)-quantitative polymerase chain reaction (qPCR). METHODS: Eighty-two bladder cancer fresh samples were collected via transurethral resection of bladder tumors. Genomic DNA was extracted from the samples, and MSRE-qPCR was performed to determine the CALN1 methylation percentage. Reverse transcription-qPCR was performed to assess the correlation between CALN1 methylation and mRNA expression. The association between CALN1 methylation percentage and clinicopathological variables of all cases and intravesical recurrence of non-muscle-invasive bladder cancer (non-MIBC) cases were analyzed. RESULTS: Of the 82 patients, nine had MIBC and 71 had non-MIBC who had not undergone total cystectomy. The median CALN1 methylation percentage was 79.5% (interquartile range: 51.1-92.6%). The CALN1 methylation percentage had a negative relationship with CALN1 mRNA expression (Spearman's ρ = - 0.563 and P = 0.012). Hypomethylation of CALN1 was associated with advanced tumor stage (P = 0.0007) and histologically high grade (P = 0.018). Furthermore, multivariate analysis revealed that CALN1 hypomethylation was an independent risk factor for intravesical recurrence in non-MIBC patients (hazard ratio 3.83, 95% confidence interval; 1.14-13.0, P = 0.031). CONCLUSION: Our findings suggest that CALN1 methylation percentage could be a useful molecular biomarker for bladder cancer.
Subject(s)
Urinary Bladder Neoplasms , Humans , Urinary Bladder Neoplasms/surgery , DNA Methylation , Cystectomy , Biomarkers , RNA, Messenger , Neoplasm Invasiveness/genetics , Neoplasm Recurrence, Local/surgerySubject(s)
DNA , Formaldehyde , Endopeptidase K , Genomics , Humans , Paraffin Embedding , TemperatureABSTRACT
BACKGROUND: MicroRNA has received considerable attention in the clinical context, and attempts are being made to use microRNA in clinical diagnosis. However, adequate quantities of microRNA required for analysis are challenging to isolate. We tested the effect of various reagents in improving microRNA extraction and compared their efficacy to that of a commercially available extraction kit (HighPure miRNA isolation kit, Roche). METHODS: We used the synthetic oligonucleotide miR-21 and formalin-fixed, paraffin-embedded (FFPE) tissue sections from colon cancer samples ( n = 10). We tested increasing volumes (100-600 µL) of 1,4-dioxane, 2-butanol, 2-propanol, acetonitrile, polyethylene glycol (PEG) 600, PEG 1000, PEG 1540, PEG 2000, tetraethylene glycol dimethyl ether (TDE), and tetrahydrofuran, instead of the binding enhancer solution provided in the kit. MiR-21 analysis was performed via stem-loop RT-qPCR using Universal ProbeLibrary probe (Roche). RESULTS: The optimum amount of each enhancement solution was 200-500 µL. We obtained ΔCp values of optimum additional volume for each solution from 1.04 to 2.50 and compared these with those obtained using the commercially available kit. PEG 1540 and 2000 produced superior reactivity with minimal addition. For FFPE tissue samples, addition of the enhancement solutions PEG 1540 and 2000 resulted in mean crossing point values of 18.15 ± 2.26 and 17.73 ± 3.26, respectively. We obtained a crossing point value of 20.56 ± 4.26 (mean ± SD) using the commercially available kit. CONCLUSIONS: The tested enhancer reagents, which are relatively readily available and easy to use, can improve microRNA extraction efficacy of a commercially available kit.
