ONCOLOGY REPORTS 45: 73, 2021 Methylation gene KCNC1 is associated with overall survival in patients with seminoma SAIPENG CHEN1,2*, LONGFEI XIAO1,2*, HUAHONG PENG3, ZHEN WANG4 and JIANBING XIE1,2 1Affiliated Hospital of Putian University, Putian, Fujian 351100;2 Department of Reproductive Medicine, Xiangyang No. 1 People's Hospital, Hubei University of Medicine, Xiangyang, Hubei 441000; 3Tianjin Institute of Urology, The Second Hospital of Tianjin Medical University, Tianjin 300211; 4Taixing People's Hospital, Taixing, Jiangsu 225400, P.R. China Received September 13, 2020; Accepted March 1, 2021 DOI: 10.3892/or.2021.8024 Abstract. The aim of the present study was to explore and and non‑seminoma. The latter can be further subdivided into verify the potential mechanism of seminoma progression. Data embryonic cell carcinoma, choriocarcinoma, yolk sac tumor or on 132 RNA‑seq and 156 methylation sites from stage II/III teratoma (2). Non‑seminomas have a high degree of differen‑ and I seminoma specimens were downloaded from The Cancer tiation but can have multiple morphological phenotypes, since Genome Atlas database. An initial filter of |fold‑change| >2 they contain both stem cells and cells that have differentiated and false discovery rate <0.05 were used to identify differ‑ to varying degrees towards somatic lineages. Seminomas have entially expressed genes (DEGs) which were associated with a lower level of differentiation and resemble primordial germ differential methylation site genes; these genes were consid‑ cells and/or intratubular germ cell neoplasia of unclassified ered potential candidates for further investigation by survival type cells (3‑5). Seminomas can be staged according to the analysis. Potassium voltage‑gated channel subfamily C degree of proliferation and metastasis. Stage I seminomas can member 1 (KCNC1) expression was verified in seminoma be completely treated by surgery and additional interventions, human tissues and three seminoma cell lines. The invasive, such as close follow‑up, drug‑assisted chemotherapy and proliferative and apoptotic abilities of the human testicular adjuvant radiotherapy (6). However, stage II/III seminomas tumor Ntera‑2 and normal human testis Hs1.Tes cell lines have a lower survival rate following surgery and postoperative were assessed following aberrant KCNC1 expression. KCNC1 radiotherapy and chemotherapy (7). Recent studies have shown was identified as a DEG, in which hypermethylation inhibited that immunotherapy and gene therapy are effective in treating its expression and it was associated with poor overall survival seminomas (8,9). Therefore, exploring the gene expression in patients with seminoma. The present results demonstrated profile of seminomas is essential to improve the understanding that KCNC1 is negatively correlated with methylation. Due to of its oncogenesis and progression. the abnormal expression of KCNC1 in seminoma cells, it was DNA methylation is an important epigenetic modifica‑ suggested that KCNC1 could be used as a diagnostic indicator tion that changes genomic expression without altering DNA and therapeutic target for the progression of seminoma. sequences. DNA methyltransferase 3α (DNMT3A), encodes a DNA methyltransferase that is thought to function in methyla‑ Introduction tion. Several studies have shown that DNA methylation plays an important role in the development and progression of tumors, Testicular germ cell tumors (TGCTs) are the most common mainly through the changes in DNA methylation patterns and solid tumors in men aged 15 to 35 years and account for >90% DNA methyltransferase expression levels (10). Evidence has of primary malignant tumors of the testes (1). TGCTs can shown that hypermethylation can inhibit the expression of be clinically classified into two main categories: Seminoma tumor‑suppressor genes, thereby promoting tumor progres‑ sion. For example, in sporadic retinoblastoma (RB), RB tumor‑suppressor gene functions may become inactivated due to methylation, with its expression levels reduced to 8%, as compared with unmethylated RB (11,12). Correspondence to: Dr Jianbing Xie, Affiliated Hospital of Putian Potassium voltage‑gated channel subfamily C member 1 University, 999 Dongzhen East Road, Licheng District, Putian, Fujian 351100, P.R. China (KCNC1) encodes a member of the family of membrane E‑mail: [email protected] proteins that mediate voltage‑dependent potassium ion permeability in excitable membranes (13). The abnormal + *Contributed equally expression and activity patterns of K channels on the surface of cancer cells can drive tumor transformation, malignant Key words: seminoma, KCNC1, methylation, K+ channels, dot blot progression and metastasis, or drug resistance, through Ca2+ activated IK or BK channels (14,15). Other studies have shown that K+ channels can affect cell proliferation and cell cycle 2 CHEN et al: LOWER KCNC1 INDICATES WORSE SURVIVAL FOR SEMINOMA PATIENTS progression in breast cancer cells (16). However, K+ channels University, Tianjin, China. The use of tumor tissue for are poorly studied in seminoma, and their role in seminoma immunohistochemistry was approved by the Second Hospital progression remains unclear. Previous studies have found that of Tianjin Medical University Ethics Committee (Tianjin, microRNA (miRNA/miR)‑199a‑3p, as a tumor‑suppressing China). The family members of the patients were informed miRNA, negatively regulates the expression of methylation that the tumor tissue removed during surgery would be used marker DNMT3A and impacts the level of glycometabolism in for further scientific research, and the approval of the family testicular germ cells, ultimately inhibiting the progression of members of the patients and their informed consent was seminomas (17,18). obtained. In the present study, the methylation sites and RNA‑seq Immunohistochemical staining of formalin‑fixed, data collected from The Cancer Genome Atlas (TCGA) were paraffin‑embedded seminoma and adjacent non‑tumor integrated to identify KCNC1 as a methylation‑regulated tissues was performed. These specimens were preserved by gene that plays an important role in the progression of malig‑ our hospital, and were approved by a pathologist prior to use. nant seminoma. Furthermore, the expression of KCNC1 in Tissues were classified according to normal testicular tissue, seminoma tissues and cell lines was verified by immuno‑ localized seminoma and metastatic seminoma (3 cases per histochemical staining, western blot analysis and reverse group). A total of 9 tissue sections from the following patho‑ transcription‑quantitative (RT‑q)PCR. In the present study, we logical groups were prepared: normal, stage I and stage II/III. hypothesized that hypermethylation can affect the expression Immunohistochemical staining using 0.4‑µm thick sections of KCNC1, thereby promoting seminoma progression. The was performed using a KCNC1 antibody (rabbit; dilution, objective was to discover novel target genes for the treatment 1:500; Abcam) and stained with hematoxylin. The staining of seminoma in order to inhibit its progression and improve was performed according to normal immunohistochemical the prognosis of patients. procedures. The human Ntera‑2 testicular tumor (NT2), normal human testis Hs1.Tes (HT) and Tcam‑2 cell lines Materials and methods (human testicular seminoma cells) were purchased from the American Type Culture Collection. They were cultured Data sourcing and analysis. RNA‑seq data from 26 patients at 37˚C with 5% CO2 in RPMI‑1640 medium (Thermo Fisher with stage I seminoma and 106 patients with stage II/III Scientific, Inc.) containing 10% FBS (Gibco; Thermo Fisher seminoma were downloaded from The Cancer Genome Atlas Scientific, Inc.) and 100 U/ml penicillin. (TCGA) database https://www.cancer.gov/tcga (19). GDCRNATools (version 1.10.1) (20) in the R package Western blot analysis and RT‑qPCR. Protease inhibi‑ (version 4.0.3) (https://cran.r‑project.org/) was used to analyze tors (Beyotime Institute of Biotechnology) were mixed differentially expressed genes (DEGs). |Fold‑change| >2 and with total protein extraction reagent (Beijing Solarbio false discovery rate (FDR) <0.05 were set as the screening Science & Technology Co., Ltd.) at a ratio of 1:100, and then thresholds. Differential methylation sites of 156 seminoma mixed with cultured cells. The protein concentration was specimens in the TCGA database were analyzed using determined using BCA. Protein samples were mixed with ELMER software package (version 2.14.0) (21). A total of loading buffer and loaded onto a 10% SDS‑PAGE for western 162 differential hypermethylation sites between stage II/III blot analysis. The protein expression levels of KCNC1, and stage I seminomas were obtained, and the regulated genes DNMT3A and GAPDH, as well as epithelial‑mesenchymal around these methylation sites were then confirmed. The transition (EMT)‑related markers and apoptotic markers were genes associated with differential hypermethylation sites were detected by western blot analysis. The antibodies used are cross‑referenced with the DEGs, and correlation analysis was listed in Table I. The primary antibody was incubated at 4˚C performed. A total of 14 methylation‑DEGs were obtained overnight, the secondary antibody was incubated at room and survival analysis was performed. The results confirmed temperature for 1 h, and ECL (Biochannel Co., Ltd.) was used that KCNC1, a gene regulated by hypermethylation, affected for exposure. the overall survival of seminoma patients. Correlation