Abnormal methylation status of FBXW10 and SMPD3, and associations with clinical characteristics in clear cell renal cell carcinoma

Jinyou Wang; Jian Li; Jun Gu; Jian Yu; Shicheng Guo; Yao Zhu; Dingwei Ye

doi:10.3892/ol.2015.3707

Abnormal methylation status of FBXW10 and SMPD3, and associations with clinical characteristics in clear cell renal cell carcinoma

Oncol Lett. 2015 Nov;10(5):3073-3080. doi: 10.3892/ol.2015.3707. Epub 2015 Sep 16.

Authors

Jinyou Wang¹, Jian Li¹, Jun Gu², Jian Yu³, Shicheng Guo⁴, Yao Zhu¹, Dingwei Ye¹

Affiliations

¹ Department of Oncology, Shanghai Medical College, Fudan University, Shanghai 200032, P.R. China ; Department of Urology, Fudan University Cancer Hospital, Shanghai 200032, P.R. China.
² State Key Laboratory of Genetic Engineering and Ministry of Education Key Laboratory of Contemporary Anthropology, School of Life Sciences, Fudan University, Shanghai 200433, P.R. China.
³ State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200240, P.R. China.
⁴ State Key Laboratory of Genetic Engineering and Ministry of Education Key Laboratory of Contemporary Anthropology, School of Life Sciences, Fudan University, Shanghai 200433, P.R. China ; State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200240, P.R. China.

Abstract

The present study aimed to evaluate the use of the 27K methylation array to investigate abnormal methylation of two genes and their associations with clinical characteristics in clear cell renal cell carcinoma (ccRCC). Six differentially-methylated genes identified using the 27K methylation array were screened in the human RCC 786-0 cell line and normal kidney tissues by bisulfite sequencing polymerase chain reaction (PCR). Differentially-methylated regions (DMRs) that were abnormally hypermethylated in the cell line were further validated in renal tumor and paired normal tissues by pyrosequencing. The correlations between DMRs and differences (methylation rate of tumor minus that of paired normal tissue) according to gender, age, tumor size, Fuhrman grade and disease stage were assessed. Gene expression prior to and following 5-Aza-2'-deoxycytidine treatment was examined using reverse transcription quantitative PCR (RT-qPCR). Two DMRs located in the FBXW10 and SMPD3 genes were found to be hypermethylated in the 786-0 cells, but not in the normal kidney tissues. Pyrosequencing results showed that the average methylation rate of FBXW10 in the cancer tissues was significantly higher compared to that in the paired normal tissues (48.78 vs. 34.62%; P<0.001). The methylation rate of SMPD3 was also higher in the cancer tissues compared with the paired normal tissues (58.98 vs. 38.66%; P<0.001). In stage T1 RCC, the methylation rate of the tumor tissue was positively correlated with the Fuhrman grade (P=0.02). The difference in methylation between the tumor and normal tissues was significantly higher in the group with high Fuhrman grade for the two genes. Furthermore, the linear correlation between methylation difference and tumor size was also confirmed (P=0.01). The RT-qPCR analysis demonstrated that SMPD3 and FBXW10 mRNA expression was significantly upregulated following 5-Aza-2'-deoxycytidine treatment. The results identified two novel DMRs located in SMPD3 and FBXW10 that were hypermethylated in the ccRCC tissue samples. The methylation profile in ccRCC could potentially provide predictive information for clinical decisions.

Keywords: DNA methylation; FBXW10; SMPD3; clear cell renal cell carcinoma.