Patients and samples
Fifty patients with ccRCC who underwent radical nephrectomy at the First Hospital of China Medical University from January 2018 to October 2018 were enrolled in the study. ccRCC tissues and corresponding para cancer tissues were obtained from renal operation and immediately stored at -80℃. The para cancer tissues were taken from a location 5 cm away from the tumor. For some patients with a large tumor with the remaining para cancer tissues being less than 5 cm, the renal tissues more than 3 cm away from the tumor were taken and pathologically proven to be cancer-free. No patients had received any preoperative adjuvant therapy, such as radiotherapy, chemotherapy, immunotherapy, targeted therapy, interventional embolization, and so on, or had a history of other malignant tumors. Demographic and clinical data of ccRCC patients are summarized in Table 1. This study was approved by the Ethics Committee of the First Hospital of China Medical University (Shenyang, China). Written informed consent was obtained from each subject.
A total of 29 fresh tissues were used to determine KDM2A mRNA levels. Total mRNAs of ccRCC and paracancer tissues were extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA.) following the manufacturer’s protocols. Reverse-transcribed cDNA synthesis was performed with BioTeke super RT Kit (BioTeke, Beijing, China) following the manufacturer’s protocols. PCR was conducted using 2×Power Taq PCR MasterMix (BioTeke, Beijing, China) and SYBR Green (BioTeke, Beijing, China). β-actin acted as an internal control. The primers were synthesized as follows: KDM2A Forward 5’-GGCAGTAGGAATCAAGGACC-3’, Reverse 5’-ACCCGACAGCAGTGAGTAGA-3’; β-actin: Forward 5’-CACTGTGCCCATCTACGAGG-3’, Reverse 5’-TAATGTCACGCACGATTTCC-3’. PCR conditions were as follows: initial denaturation at 94℃ for 5 min, followed by 40 cycles of 94℃ for 15 sec, 60℃ for 20 sec, and 72℃ for 30 sec. Each reaction was set up 3 times and the expression level of KDM2A was quantified using the 2−△△Ct method.
All tissues were fixed in 4% polyoxymethylene, embedded in paraffin, and sectioned at 4 μm. Then, the slides were treated with xylene to remove the paraffin, followed by hydration with ethanol and the addition of EDTA for antigen retrieval. Endogenous peroxidase blocker solution was added to incubate for 10 min, and the sections were rinsed with PBS 3 times. To avoid nonspecific binding, normal goat serum was added to block tissue collagen for 10 min. Sections were incubated with KDM2A monoclonal antibody (1:250; Abcam, Cambridge, MA, USA) for 1 h at room temperature. After washing 3 times with PBS, the sections were incubated with biotinylated secondary goat anti-rabbit antibody, and then with streptavidin-biotin peroxidase for 10 min each. Finally, the diaminobenzidine (DAB) solution was used to stain the sections, which were then counterstained with hematoxylin.
The KDM2A IHC score was determined by both the intensity and percentage of cellular staining. The staining intensity was divided into scores of 0 (negative), 1 (mild), 2 (moderate), 3 (strong), and the percentage was classified as 1 (0-25%), 2 (26-50%), 3 (51-75%), and 4 (>75%). The intensity and percentage were multiplied to calculate the total IHC staining score, which was assigned as negative staining (-, 0), mild staining (+, 1-4), moderate staining (++, 5–8), severe staining (+++, 9–12). An IHC score ≥ 5 was defined as high expression, while a score of less than 5 was defined as low expression. Two independent observers were employed to assess and examine immunostaining.
Survival data from TCGA was obtained to clarify the prognostic value of KDM2A in ccRCC. They were submitted to OncoLnc (http://www.oncolnc.org/) and Kaplan-Meier curve was plotted for prognostic analysis.
Statistical analysis was performed using SPSS version 23. Quantitative data were expressed as the mean ± standard deviation (SD), and counting data were represented by the number and rate. The significance of differences in expression levels of KDM2A between ccRCC tissues and para cancer tissues was calculated by independent sample t-test, χ2 test, or Fisher’s exact test, as appropriate Spearman’s rank correlation test was used to analyze the association between KDM2A expression and clinical parameters of ccRCC patients. All P values were two-sided, and values of less than 0.05 were considered statistically significant.