特异性U1snRNA和Morpholino对SCN1A基因剪接突变的修复机理探讨

发布时间：2018-06-04 22:29

本文选题：SCN1A基因突变 + 剪接　；参考：《广州医科大学》2014年硕士论文

【摘要】：【目的】选取热性惊厥相关性癫痫患者SCN1A基因突变，通过生物信息学软件分析其对剪切的影响，构建mini-gene，进行体外剪接分析；并探讨特异性U1snRNA及Morpholino修复异常剪接的机理。【对象和方法】选取本实验室筛查出的热性惊厥相关性癫痫患者SCN1A基因突变(c.473+5GA,c.909AG, c.3705+1GT)为研究对象，用Human Splicing Finder2.4软件进行预测c.473+5GA和c.909AG突变体的异常剪接情况（其中c.3705+1GT的异常剪接情况在本课题组既往研究已明确）。构建mini-gene（pTARGET-Exon2-3-4-5，pTARGET-Exon5-6-7-8）转染HEK293细胞，提取总RNA，进行RT-PCR,分析异常剪接的情况。根据上述三个突变位点设计与剪接供体位点完全互补或与其附近内含子区，外显子区多个靶位点互补的特异性U1snRNA表达载体；与野生型或突变型mini-gene（1:1）共转染HEK293细胞，，提取总RNA，进行RT-PCR，分析正常与异常转录本比例，比较特异性U1snRNA修复异常剪接的效果。同时对突变c.909AG设计与其新激活的剪接供体位点互补的Morpholino寡核苷酸，分析对异常剪接的修复效果，并与其U1snRNA靶向修复的效果对比。每组实验重复三次，获得均值，计量资料是由均数±标准差（X±S)表示，使用SPSS16.0软件包进行统计分析。两样本均数比较采用t检验。以P 0.05具有统计学意义。【结果】 1. SCN1A基因突变体外剪切分析结果 PEFS+患者c.473+5GA突变体异常转录本较正常的缺少98bp碱基，即SCN1A基因的2号外显子5'端8bp碱基和全3号外显子的缺失。FS+患者c.909AG突变体异常转录本较正常的缺少60bp碱基，即6号外显子5'端60bp碱基缺失。两种异常剪切结果与软件预测结果一致。 2.特异性U1snRNA修复异常剪切的效果（1） c.473+5GA的mini-gene突变体，表达正常转录本占总转录本（异常转录本和正常转录本的和）的比例为35.37%±3.29%。与特异性U1snRNA(E3Mu,E3+1, E3+9, E3+16)共转染后，正常转录本比例增加至59.88%±4.46%，54.94%±1.97%，100%，100%。它们与单纯突变体转染时正常转录本比例相比，均有统计学差异(p0.05)。（2）c.909AG的mini-gene突变体，表达正常转录本占总转录本比例为30.34%±2.71%。与特异性U1snRNA(E6+1, E6+9, E6+16, E6-9, E6-18)共转染后，正常转录本比例分别为92.85%±2.08%,100%,100%,27.47%±1.80%,29.83%±3.54%。与单纯突变体的正常转录本比例相比，E6+1, E6+9, E6+16有统计学差异(p0.05)；E6-9, E6-18无统计学差异(p0.05)。（3）c.3705+1GT的突变体mini-gene（产生两个异常转录本，即SCN1A基因的18号外显子5'端缺失49bp碱基或者18号内含子5'端保留20bp碱基），表达大片段异常转录本占总转录本的比例为37.53%±2.27%。与特异性U1snRNA(E18Mu, E18+1, E18+9, E18+16)共转染后，表达大片段异常转录本比例分别为55.49%±1.38%，64.66%±3.64%，46.39%±2.14％，80.29%±3.51%，与单纯突变体的相比，有统计学差异(p0.05)，但无正常转录本的表达。 3. Morpholino寡核苷酸的修复异常剪切的效果 c.909AG的mini-gene突变体，表达正常转录本占总转录本比例为30.75%±2.25%，与Morpholino（终浓度分别为10uM，50uM）共转染后，正常转录本比例分别为41.64%±1.94%，64.54%±2.47%，27.24%±2.72%；与单纯突变体的相比，有统计学差异(p0.05)。【结论】 1.SCN1A基因c.437+5GA和c.909AG突变，可能通过影响剪切与疾病的发生相关。 2.特异性U1snRNA对不同位置的突变修复效果不一样，其中剪接供体位点高度保守区+1位置的突变修复效果最差；结合于不同靶位点的特异性U1snRNA修复效果不同，结合于内含子区的修复效果较好。 3.特异性U1snRNA和Morpholino对异常剪切都有修复作用，但U1snRNA的修复效果较好。
[Abstract]:[Objective]
The SCN1A gene mutation in epileptic patients with febrile seizures was selected, and the effects on shear were analyzed by bioinformatics software. The mini-gene was constructed and the splicing was analyzed in vitro, and the mechanism of abnormal splicing of specific U1snRNA and Morpholino was discussed.
[object and method]
The SCN1A gene mutation (c.473+5GA, c.909AG, c.3705+1GT) in patients with febrile convulsion related seizures (c.909AG, c.3705+1GT) was selected as the research object. The abnormal splicing of c.473+5GA and c.909AG mutants was predicted with Human Splicing Finder2.4 software (in which the abnormal splicing of c.3705+1GT was clearly defined in our previous research group). Mini-gene (pTARGET-Exon2-3-4-5, pTARGET-Exon5-6-7-8) was transfected into HEK293 cells, total RNA was extracted, RT-PCR was analyzed, and abnormal splicing was analyzed.
According to the above three mutation sites, the specific U1snRNA expression vector was complementation with the splicing site or its nearby introns, and the target loci of the exons were complementing. The HEK293 cells were co transfected with the wild type or mutant mini-gene (1:1), and the total RNA was extracted, and the ratio of normal to abnormal transcriptional transcript was analyzed, and the ratio of normal to abnormal transcriptional transcript was comparatively specific. The effect of abnormal splicing was repaired by sexual U1snRNA. At the same time, the Morpholino oligonucleotides complementing the mutation c.909AG design with the newly activated splice donor sites were analyzed and compared with the effect of the U1snRNA targeted repair. Each group of experiments repeated three times and obtained the mean value. The measurement data were mean standard deviation (X + S) table. SPSS16.0 software package was used for statistical analysis. Two samples were compared with t test. P 0.05 was statistically significant.
[results]
The results of in vitro shear analysis of 1. SCN1A gene mutations
The abnormal transcriptional transcript of the c.473+5GA mutant in PEFS+ patients is less than the normal 98bp base, that is, the 8bp base of the SCN1A gene 2 exon 5'terminal 8bp base and the total 3 exon deletion.FS+ patient c.909AG mutant abnormal transcriptional transcript is less than normal 60BP base, that is, the 60BP base deficiency at the 5' end of exon 6. Two abnormal shear results and software prediction results. Agreement.
The effect of 2. specific U1snRNA in the repair of abnormal shear
(1) the mini-gene mutant of c.473+5GA, which expressed the proportion of normal transcriptional transcripts in the total transcriptional transcript and normal transcript, was 35.37% + 3.29%. and specific U1snRNA (E3Mu, E3+1, E3+9, E3+16) Co transfected, and the normal transcriptional ratio increased to 59.88% + 4.46%, 54.94% + 1.97%, 100%, and 100%., and they were transfected with the simple mutant. The proportion of frequent transcripts was statistically different (P0.05).
(2) the mini-gene mutant of c.909AG, the normal transcriptional transcript was co transfected with the total transcriptional ratio of 30.34% + 2.71%. and specific U1snRNA (E6+1, E6+9, E6+16, E6-9, E6-18), the normal transcriptional ratio was 92.85% + 2.08%, 100%, 100%, 27.47% + 1.80%, 29.83% + 3.54%. compared to the normal transcriptional ratio of the simple mutant, E6+1, E6+9. 6+16 had statistical difference (P0.05); E6-9 and E6-18 had no statistical difference (P0.05).
(3) the mutant mini-gene of c.3705+1GT (producing two abnormal transcripts, namely the 5'terminal deletion 49bp base of the exon 18 of the SCN1A gene or the 20bp base in the 5' end of the intron 18), expressed the proportion of the large fragment of the abnormal transcriptional book in the total transcript of 37.53% + 2.27%. and the specific U1snRNA (E18Mu, E18+1, E18+9, and E18+9). The proportion of abnormal transcripts was 55.49% + 1.38%, 64.66% + 3.64%, 46.39% + 2.14% and 80.29% + 3.51% respectively. Compared with the simple mutant, there were statistical differences (P0.05), but there was no normal transcriptional transcript.
Effect of 3. Morpholino oligonucleotide repair on abnormal shearing
The normal transcriptional version of the mini-gene mutant of c.909AG was 30.75% + 2.25%, and the normal transcriptional ratio was 41.64% + 1.94%, 64.54% + 2.47% and 27.24% + 2.72%, respectively, with Morpholino (final concentration 10uM, 50uM). Compared with the simple mutant, there was a statistically significant difference (P0.05).
[Conclusion]
The mutation of c.437+5GA and c.909AG of 1.SCN1A gene may be related to the occurrence of disease through shear.
The effect of 2. specific U1snRNA on mutation repair at different locations is different, in which the mutation repair effect of the splice donor site at the highly conserved +1 location is the worst, and the effect of the specific U1snRNA repair combined with the different target loci is different, and the repair effect is better in the intron in the intron.
3. specific U1snRNA and Morpholino can repair abnormal shear, but U1snRNA has better repair effect.
【学位授予单位】：广州医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R742.1

【参考文献】