CYP17A1通过介导DNA去甲基化对胶质瘤细胞生物学特性的影响及相关机制  

Effect of CYP17A1 on the biological characteristics of glioma cells and related mechanisms by mediating DNA demethylation

作　　者：郭斯檾[1] 梁庆新[1] 贾若飞 GUO Si-qing;LIANG Qing-xin;JIA Ruo-fei(Department of Neurosurgery,Foshan Hospital of Traditional Chinese Medicine,Foshan Guangdong 528000,China)

机构地区：[1]佛山市中医院神经外科,广东佛山528000

出　　处：《临床和实验医学杂志》2021年第14期1467-1470,共4页Journal of Clinical and Experimental Medicine

基　　金：广东省省级科技计划项目(编号:2016A020226052);佛山市科技创新项目自筹经费类科技计划项目(编号:1920001000655)。

摘　　要：目的探讨细胞色素P450c17a酶(CYP17A1)通过介导DNA去甲基化对胶质瘤细胞生物学特性的影响,并分析其相关机制。方法人胶质瘤细胞U251分为干预组(先用DHEA处理48 h,再用TMZ预处理48 h)和阴性对照组(正常培养,不进行任何干预),各6个复孔;以正常脑组织坏死细胞(非胶质细胞)为正常组,正常培养,不进行任何干预。通过聚合酶链式反应(PCR)实验,蛋白质印迹实验对细胞中CYP17A1 mRNA及蛋白表达量进行测定;利用甲基化特异性PCR法检测CYP17A1基因在的甲基化状态;通过MTT及流式细胞仪对细胞增殖情况进行测定;通过细胞侵袭实验对细胞侵袭及转移情况进行测定。通过细胞集落形成试验检测CYP17A1的增殖能力。结果提取总RNA的完整性,28S条带比18S处条带亮2倍左右,后面未见有拖带痕迹。干预组与阴性对照组CYP17A1 mRNA的相对表达量(0.811±0.047、0.507±0.025)均显著高于正常组(0.020±0.001),而干预组显著低于阴性对照组,差异均有统计学意义(P<0.05)。干预组CYP17A1蛋白(0.405±0.031)显著低于阴性对照组(0.783±0.032),差异有统计学意义(P<0.05),该蛋白在正常组中不表达。CYP17A1基因在正常细胞中未发生甲基化,干预组中CYP17A1基因甲基化发病率[(43.93±8.93)%]显著低于阴性对照组[(89.03±10.02)%],差异有统计学意义(P<0.05)。干预组CYP17A1的细胞增殖速率以及细胞侵袭、迁移率分别为(38.97±11.32)%、(245.19±18.71)个、(69.09±6.53)%,均显著低于阴性对照组[(78.09±10.21)%、(587.36±43.25)个、(80.49±8.76)%],差异均有统计学意义(P<0.05)。干预96 h后,阴性对照组和干预组细胞集落形成能力[(62.71±15.25)%、(54.16±14.82)%]均显著高于正常组[(18.17±6.35)%],干预组显著低于阴性对照组,差异均有统计学意义(P<0.05)。结论CYP17A1可通过介导DNA去甲基化从而对胶质瘤细胞的增殖、侵袭和转移能力产生抑制作用,并进一步抑制细胞细胞集落形成能力�Objective To investigate the effect of Cytochrome P450c17(CYP17A1)on the biological characteristics of glioma cells by mediating DNA demethylation,and analyze its related mechanisms.Methods Human glioma cells U251 were divided into intervention group(treated with DHEA for 48h,and then pretreated with TMZ for 48h)and negative control group(normal culture without any intervention),each with 6 replicate holes,and necrotic cells of normal brain tissue(non-glial cells)were regarded as the normal group,and they were cultured normally without any intervention.The expression of CYP17A1 mRNA and protein in cells was determined by PCR and Western blotting,the methylation status of CYP17A1 gene was detected by MSP,the proliferation of cells was measured by MTT and flow cytometry,and the invasion and metastasis of cells were determined by cell invasion experiment.The proliferation of CYP17A1 was detected by colony forming assay.Results The integrity of the total RNA extracted.The 28S band is about 2 times brighter than the 18S band,and there is no trace of dragging behind.The relative expression of CYP17A1 mRNA in the intervention group and the negative control group(0.811±0.047,0.507±0.025)was significantly higher than those in the normal group(0.020±0.001),while the intervention group was significantly lower than the negative control group,and the differences were statistically significant(P<0.05).The CYP17A1 protein(0.405±0.031)in the intervention group was significantly lower than that in the negative control group(0.783±0.032),and the difference was statistically significant(P<0.05).The protein was not expressed in the normal group.CYP17A1 gene did not undergo methylation in normal cells,the incidence of CYP17A1 gene methylation in the intervention group[(43.93±8.93)%]was significantly lower than that of the negative control group[(89.03±10.02)%],the difference was statistically significant(P<0.05).The cell proliferation rate,cell invasion and migration rate of CYP17A1 in the intervention group were(38.97±11.32)

关 键 词：胶质瘤细胞 CYP17A1 甲基化 增殖 侵袭 转移 

分 类 号：R739.41[医药卫生—肿瘤]