胞嘧啶-磷酸-鸟嘌呤基序岛区甲基化对微小RNA-129-2表达调控及肾癌细胞增殖和克隆形成的影响

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

目的 观察微小RNA（miRNA,miR）-129-2上游胞嘧啶-磷酸-鸟嘌呤基序（CpG）岛区甲基化对其在肾癌中的表达调控,并探讨对肾癌细胞增殖和克隆形成能力的影响.方法 应用甲基化特异性聚合酶链反应（MSP）和亚硫酸盐处理测序法（BSP）检测miR-129-2上游CpG岛区的甲基化状态,探针法实时荧光定量反转录-聚合酶链反应（Taqman-RT-PCR）检测miR-129-2的表达,噻唑蓝（MTT）法、平板克隆形成实验分别检测去甲基化对肾癌细胞增殖和克隆形成能力的影响.结果 MSP显示在肾癌组织中miR-129-2是高甲基化状态,同时BSP结果显示经去甲基化试剂处理后的肾癌细胞株（786-O、ACHN、Caki-1和796-P）中miR-129-2的甲基化程度降低[（66.16±6.20）％比（85.63±6.27）％、（66.20±6.55）％比（86.74±4.42）％、（59.00±4.58）％比（76.85±9.40）％、（63.13±6.07）％比（80.32±8.39）％;t=3.824,4.498、2.956、2.874;P=0.019、0.011、0.042、0.045];表达水平显著升高,与对照组比较分别为（0.107 ±0.001比0.059±0.005、0.084±0.007比0.027±0.005、0.173±0.006比0.151±0.050、0.114±0.006比0.079±0.004）;t =9.793、11.897、3.093、7.566;P =0.001、0.000、0.036、0.002）;与各对照组比较,786-0、ACHN和769-P细胞株在去甲基化试剂处理后对细胞增殖能力（0.97±0.16比1.42±0.13、1.34±0.09比1.76±0.06、0.92±0.04比1.16±0.07;t=3.804、6.329、5.220;P=0.019、0.003、0.006）和克隆形成能力[（69±3）个比（102±6）个、（57±5）个比（78±6）个、（91±5）个比（112±6）个;f=8.498、4.571、5.157;P=0.001、0.010、0.007]两个方面均减弱.结论 miR-129-2的表达受DNA甲基化调控,且与肾癌细胞的增殖和克隆形成能力密切相关.

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 （MTF） 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±0.16vs.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.