MEF2C基因启动子区碱基突变对转录活性的影响  

Effect of base mutation in MEF2C promoter region on transcription activity

作　　者：邱进[1] 刘辉[2] 热孜宛古丽.伊敏 袁芳[1] 许瑞[1] 马玲[3] 霍强[4] 周勇[1] QIU Jin LIU Hui Reziwanguli Yimin YUAN Fang XU Rui MA Ling HUO Qiang ZHOU Yong(Department of Biology, College of Preclinical Medical, Xinjiang Medzcal University, Urumqi 830011, China Institute of Clinical Medicine, the First Affiliated Hospital, Xinjiang Medical University, Urumqi 830054 China Department of Nutrition and Food Hygiene,College of Public Health, Xinjiang Medical University, Urumqi 830011, China Department of Cardiac Surgery, the First Affiliated Hospital, Xinjiang Medical University, Urumqi 830054, China)

机构地区：[1]新疆医科大学基础医学院生物学教研室,乌鲁木齐830011 [2]新疆医科大学第一附属医院临床医学研究院,乌鲁木齐830054 [3]新疆医科大学公共卫生学院营养与食品卫生教研室,乌鲁木齐830011 [4]新疆医科大学第一附属医院心脏外科,乌鲁木齐830054

出　　处：《新疆医科大学学报》2017年第5期606-611,616,共7页Journal of Xinjiang Medical University

基　　金：国家自然科学基金(30901474)

摘　　要：目的研究MEF2C基因启动子突变对其转录活性的影响及其突变可能的致病机制。方法采用聚合酶链式反应,以对照组基因组DNA为模板,获得野生型MEF2C基因启动子区DNA片段(-939^+531bp)(1 470bp);利用分子克隆技术将目的片段连接到含有荧光素酶报告基因的pGL3-Basic载体上,构建野生型MEF2C启动子报告基因载体pGL3-Basic-MEF2C-WT;利用点突变技术,构建突变型MEF2C启动子报告基因载体pGL3-Basic-MEF2C-M1(M1点突变类型:-293插入G突变)、pGL3-Basic-MEF2C-M2(M2点突变类型:-160插入G,C>T突变);采用琼脂糖凝胶电泳、双酶切、基因测序方法验证上述3种载体是否构建成功;利用脂质体瞬时转染技术将pGL3-Basic-MEF2C-WT、pGL3-Basic-MEF2C-M1、pGL3-Basic-MEF2C-M2载体分别体外转染HEK-293T细胞,标记为WT组、M1组、M2组,将pGL3-Basic空载体转染HEK-293T细胞标记为对照组、将相同条件下培养未转染载体的细胞标记为空白组,采用双荧光素酶报告基因检测系统检测各组相对荧光素酶活性(relative luciferase activity RLA),比较各组载体的转录活性。数据用SPSS18.0软件进行单因素方差分析(ANOVA)处理,运用启动子区生物信息预测软件对各组启动子区序列可能的转录因子结合位点及CpG岛进行预测,并分析比较。结果成功构建了含野生型、M1型、M2型的MEF2C基因启动子区的荧光素酶报告基因载体pGL3-Basic-MEF2C-WT、pGL3-Basic-MEF2C-M1、pGL3-Basic-MEF2C-M2;空白组、对照组、WT组、M1组、M2组的RLA分别为(0.83±0.35)、(2.28±0.36)、(24.17±0.50)、(29.65±2.40);M1组和M2组的RLA水平高于WT组,分别是野生型的10.6倍、13倍,差异具有统计学意义(P<0.05);转录因子结合位点预测发现正常MEF2C基因启动子片段上-356^-108bp存在13个可能的位点,M1突变导致1个新的转录因子结合位点JCV-repeated-sequence生成,M2突变导致1个新的转录因子结合位点Sp1生成;预测发现正常MEF2C基因启动子片段上�Objective To study the effect of MEF2 Cgene promoter mutation on its transcriptional activity,and to explore the possible pathogenic mechanism of these mutations.Methods Polymerase chain reaction（PCR）was used to obtain DNA fragment of the wild type MEF2 C gene promoter in the control group DNA（-939-＋531bp）（1 470bp）,Molecular cloning technique was used to connect the target fragment to the pGL3-Basic vector containing luciferase reporter gene,and then to construct the wild-type MEF2 C promoter reporter vector pGL3-Basic-MEF2C-WT.The technology of site-directed mutagenesis was used to construct the mutant MEF2 C promoter reporter gene vector pGL3-Basic-MEF2C-M1（M1 point mutation type：-293 insert G）,pGL3-Basic-MEF2C-M2（M2mutation type：-160 insert G,C〉T）.agarose gel electrophoresis,double enzyme digestion and gene sequencing were used to verify whether the three vectors were constructed successfully.PGL3-Basic-MEF2C-WT,pGL3-Basic-MEF2C-M1 and pGL3-Basic-MEF2C-M2 vectors were transfected into HEK-293 Tcells in vitro by transient transfection technique with liposome,and termed as WT group,M1 group and M2 group respectively.The pGL3-Basic vector was transfected into the HEK-293 Tcells named as the control group.agroup of cells with the same culture condition were named as blank group.the luciferase activity（relative luciferase activity RLA）of each group was detected by dual luciferase reporter assay system,and then the transcriptional activity of each group was compared.Difference between data was analyzed by single factor analysis of variance（ANOVA）with SPSS18.0software version.the prediction of transcription factor binding sites and CpG islands in the promoter regions of each group was performed using the promoter prediction software.Resuls Luciferase reporter gene vector containing MEF2 Cgene promoter region of wild type,M1 type and M2 type was successfully constructed,named as pGL3-Basic-MEF2C-WT,pGL3-Basic-MEF2C-M1,pGL3-Basic-MEF2CM2.The RLA of blank,control,WT,M1 and M2group wer

关 键 词：肌细胞增强因子2 启动子突变 单纯性先天性心脏病 双荧光素酶报告基因系统 维吾尔族人群 

分 类 号：R394[医药卫生—医学遗传学]