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  • Clinical specimens were obtained from 135 total patients with bone tumors, treated in our institutes between 2007 and 2014. Cases included 63 male (46.7%) and 72 female (53.3%) patients (Table 1). The histological types included GCTB (n = 18), chondrosarcoma (n = 17), osteosarcoma (n = 16), osteochondroma (n = 12), and other various types. The specimens were obtained by using core-needle biopsy, incisional biopsy, or surgical resection. The diagnosis was confirmed histopathologically according to the World Health Organization classification [14]. Two experienced pathologists independently diagnosed each case. In cases that were difficult to diagnose, the pathologists reviewed and discussed the cases carefully and reached a consensus. Written informed consent was obtained from all patients for participation in this study, and this study was approved by the Ethics Committee of School of Medicine, Niigata University. Table data removed from full text. Table identifier and caption: 10.1371/journal.pone.0154680.t001 List of histological types and numbers of clinical specimens. First, total RNA was extracted from frozen samples using ISOGEN (Nippon Gene, Tokyo, Japan). The yield and purity of RNA were determined based on spectrophotometric measurements of the ratio of UV absorbance at 260 and 280 nm. First strand cDNA was synthesized from the total RNA by using PrimeScript RT Reagent Kit (TaKaRa Bio, Shiga, Japan). Quantitative real-time PCR was performed using SYBR Premix EX Taq II (Tli RNaseH Plus; TaKaRa, Shiga, Japan), and the results were analyzed using the Thermal Cycler Dice Real Time System TP800 (TaKaRa, Shiga, Japan). The primer sequences used were as follows: primer pairs used for human RANKL, 5′-GCCTTTCAAGGAGCTGTGCAA-3′, (forward) and 5′-ATCTAACCATGAGCCATCCACCAT-3′ (reverse); RANK, 5′-GCCATCATCTTTGGCGTTTG-3′ (forward) and 5′-CAAAGTTTGCCGTGTGTGTACTG-3′ (reverse); OPG 5′-CAATTTGCCTGGCACCAAAG-3′ (forward) and 5′-AGGTGAGGTTAGCATGTCCAATGT-3′ (reverse); GAPDH, 5′- GCACCGTCAAGGCTGAGAAC-3′ (forward) and 5′-TGGTGAAGACGCCAGTGGA-3′ (reverse). The gene copy numbers for RANKL, RANK and OPG were calculated using a standard curve that was constructed using the Saos2 cell line. Moreover, quantitative mRNA expression levels were calculated by normalizing against RPMI 8226, a human multiple myeloma cell line that expresses RANKL. We compared the median of the relative expression level in all histological types of tumors by using a box plot for expression of RANKL, RANK, and OPG. For histological types in which only one case was sampled, we used only a dot plot. RANKL expression was valued by using immunohistochemistry. Among 135 patients, 66 specimens were evaluable for RANKL expression. Histological types determined in these expreriments are shown in Table 2. Formalin-fixed, paraffin-embedded (FFPE) tissue sections (4-um) had the paraffin removed and were hydrated. The sections were heated with an autoclave (121°C for 20 minutes) in histofine antigen retrieval buffer, pH 9 (Nichirei Bioscience, Tokyo, Japan), and blocked against endogenous peroxidase activity and staining reagent. Tissue sections were incubated overnight at 4°C with rabbit anti-human RANKL antibody (ab9957; Abcam, Cambridge, UK). Primary antibodies were visualized by using the histofine Simple Stain MAX-PO (MULTI) kit (Nichirei Bioscience) and 3, 3’-diaminobenzidine (Simple Stain DAB, Nichirei Bioscience. The sections were counterstained in Mayer’s hematoxylin. Table data removed from full text. Table identifier and caption: 10.1371/journal.pone.0154680.t002 List of histological types and numbers assessed by immunohistochemistry. Statistical analysis was conducted with the Statistical Package for the Social Sciences (SPSS Inc. Chicago, Illi-nois, USA) version 21.0. We performed the statistical analysis for the 15 histological types for which there were more than two cases. As the data were not normally distributed as determined by using Shapiro-Wilk test, they were analyzed with a post-hoc multiple comparison. The significance level was set at P < 0.05.
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