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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基因启动子片段上存在2个CpG岛,2个突变组预测没有发现CpG岛数目的变化,但位置发生变化。结论 MEF2C基因启动子区M1突变、M2突变均能提高转录活性,这2种突变导致启动子区转录因子结合位点数目、CpG岛位置的改变,这些变化可能与新疆维吾尔族单纯性先天性心脏病的发病机制有关。
Objective To study the effect of MEF2C gene promoter mutation on its transcriptional activity and its possible mechanism of mutation. Methods DNA fragment (-939 ~ + 531 bp) (1 470 bp) in the promoter region of wild-type MEF2C gene was obtained by polymerase chain reaction (PCR) using the genomic DNA of the control group as a template. The target fragment was ligated to a DNA fragment containing fluorescein MEF2C-M1 (M1) was constructed based on the pGL3-Basic vector of the enzyme reporter gene and the wild type MEF2C promoter reporter gene vector pGL3-Basic-MEF2C-WT. Point mutation type: -293 insert G mutation), pGL3-Basic-MEF2C-M2 (M2 point mutation type: -160 insert G, C> T mutation); using agarose gel electrophoresis, double enzyme digestion, gene sequencing method validation The three kinds of vectors were constructed successfully. The vectors of pGL3-Basic-MEF2C-WT, pGL3-Basic-MEF2C-M1 and pGL3-Basic-MEF2C-M2 were transfected into HEK-293T cells in vitro using lipofectamine. The cells transfected with empty vector pGL3-Basic and HEK-293T cells were marked as control group, and the untransfected cells under the same conditions were labeled as blank group and labeled with dual luciferase Gene detection system to detect the relative luciferase activity of each group (relative luciferase act ivity RLA) to compare the transcriptional activity of each group of vectors. Data were analyzed by one-way analysis of variance (ANOVA) using SPSS18.0 software. The predicted transcription factor binding sites and CpG islands of promoter regions in each group were predicted by the software of promoter bioinformatics prediction and analyzed. Results The luciferase reporter gene vectors pGL3-Basic-MEF2C-M1, pGL3-Basic-MEF2C-M2 containing the MEF2C gene promoter region of wild type, M1 type and M2 type were successfully constructed. The RLA levels in the blank group, the control group, the WT group, the M1 group and the M2 group were (0.83 ± 0.35), (2.28 ± 0.36), (24.17 ± 0.50) and (29.65 ± 2.40) Which was 10.6-fold and 13-fold higher than that in WT group, respectively (P <0.05). Prediction of transcription factor binding sites showed that there were 13 possible -356 ~ -108bp fragments in the normal MEF2C gene promoter fragment M1 mutation resulted in the generation of a new transcription factor binding site JCV-repeated-sequence. M2 mutation resulted in the generation of a new transcription factor binding site Sp1. Two MEF2C gene promoter fragments were predicted CpG islands, two mutations predicted no change in the number of CpG islands, but the location changed. Conclusions Mutations of M1 and M2 in MEF2C gene promoter both increase the transcriptional activity. These two mutations lead to changes in the number of transcription factor binding sites and CpG island in the promoter region, which may be related to the simple Uygur congenital heart The pathogenesis of the disease.