Regulation of microRNA-129-2 expression by cytosine-phosphoric acid-guanine motif island methylation and its effect on the proliferation and colony formation in renal cell carcinoma

Abstract

Objective To investigate the impact of methylation of the cytosine-phosphoric acid-guanine motif (CpG) island upstream of microRNA (miRNA, miR)-129-2 on regulation of expression and its effect on the proliferation and colony formation in renal cell carcinama. Methods The methylation specific polymerase chain reaction (MSP) and the bisulfite sequencing PCR (BSP) were implemented to detect CpG island methylation of miR-129-2 in the upstream region. Taqman real-time fluorescence quantitative polymerase chain reaction (Taqman-RT-PCR) was applied for identifying of miR-129-2 expression. The proliferation and colony formation of renal cell lines were detected by Thiazole blue (MTT) and colony formation assay. Results MSP results revealed that miR-129-2 was hypermethylated in tumor tissues, while BSP showed a decrease in the degree of methylation of miR-129-2 in renal cell lines (786-O, ACHN, Caki-1 and 796-P) treated with demethylating agents [(66.16±6.20)% vs. (85.63±6.27)%, (66.20±6.55)% vs. (86.74±4.42)%, (59.00±4.58)% vs. (76.85±9.40)%, (63.13±6.07)% vs. (80.32±8.39)%; t=3.824, 4.498, 2.956, 2.874; P=0.019, 0.011, 0.042, 0.045]. Compared with control group each cell line, demethylating agent elevated miR-129-2 expression (0.107±0.001 vs. 0.059±0.005, 0.084±0.007 vs. 0.027±0.005, 0.173±0.006 vs. 0.151±0.050, 0.114±0.006 vs. 0.079±0.004; t=9.793, 11.897, 3.093, 7.566; P=0.001, 0.000, 0.036, 0.002). Compared with control group above, the proliferation (0.97±0.16 vs. 1.42±0.13, 1.34±0.09 vs. 1.76±0.06, 0.92±0.04 vs. 1.16±0.07; t=3.804, 6.329, 5.220; P=0.019, 0.003, 0.006) and colony formation [(69±3) cells vs. (102±6) cells, (57±5) cells vs. (78±6) cells, (91±5) cells vs. (112±6) cells; t=8.498, 4.571, 5.157; P=0.001, 0.010, 0.007] significantly were inhibited in 786-O, ACHN and 769-P cell lines that were observed after treated with demethylating agent. Conclusion The expression of the miR-129-2 is regulated by the DNA methylation and closely related to the proliferation and colony formation ability. Key words: MicroRNA-129-2; Methylation; Renal cell carcinama; Proliferation; Colony formation