Perrault综合征HARS2基因c.349G>A、c.908T>C突变对细胞线粒体功能的影响

Effects of c. 349G>A and c. 908T>C mutations in HARS2 gene of Perrault syndrome on mitochondrial function

摘要:

目的 探讨HARS2基因c.349G>A和c.908T>C两个位点的突变对细胞和线粒体功能的影响，以期揭示HARS2新突变位点的致病机制。方法 构建含野生型、c.349G>A突变型、c.908T>C突变型HARS2基因的重组表达载体，使用慢病毒包装系统将质粒转染至HEK 293T细胞中，通过嘌呤霉素筛选获得稳定转染野生型、突变型HARS2基因的细胞系，同时建立稳定转染空病毒载体的细胞系作为对照，对野生组、c.349G>A突变组、c.908T>C突变组和对照组细胞的线粒体功能和细胞增殖能力进行研究。结果 重组质粒在预期位点发生了突变，重组载体携带的EGFP绿色荧光蛋白稳定表达；两突变组细胞增殖活力较野生组下降，72 h吸光度有显著性差异(P<0.05)；c.349G>A突变组MTCO2蛋白表达量较对照组和野生组均有下降(P<0.05)；c.349G>A和c.908T>C突变组的线粒体ATP相对细胞总ATP产率较对照组均有降低(P<0.05)；c.349G>A突变组线粒体活性氧自由基（reactive oxygen species， ROS）水平较对照组明显增加(P<0.05)；两突变组线粒体膜电位（mitochondrial membrane potential，MMP）较野生组下降(P<0.01)。结论 HARS2基因c.349G>A、c.908T>C突变细胞的线粒体存在一定的呼吸功能缺陷，突变细胞增殖能力下降，可能是HARS2基因突变致Perrault综合征(Perrault syndrome, PRLTS)的细胞学机制。

Abstract:

Objective To investigate the effect of c.349G>A and c. 908T>C mutations in HARS2 gene on mitochondrial and cell function, and to reveal the pathogenic mechanism of HARS2 mutation. Methods Recombinant expression vectors containing wild-type, c.349G>A mutant and c.908T>C mutant HARS2 were constructed. The plasmid was transfected into HEK 293T cells using lentivirus packaging system. Stably transfected cell lines were screened using puromycin. Meanwhile, the cell line transfected with empty virus vector was established as the control. The mitochondrial function and cell proliferation of HEK 293T cell lines in wild group, two mutant groups and control group were studied. Results The recombinant plasmid was mutated at the desired site, and the EGFP protein carried by the recombinant vector was expressed stably; The cell proliferation activity of the two mutant groups was lower than that of the wild group, and the absorbance at 72 h was significantly different (P<0.05); MTCO2 protein expression in c.349G>A mutant group was lower than that in control group and wild group (P<0.05); Compared with the control group, the mitochondrial ATP relative to total cellular ATP production was decreased in both mutation groups (P<0.05); The level of mitochondrial reactive oxygen species (ROS) in the c.349G>A mutant group was increased compared with the control group (P<0.05); The mitochondrial membrane potential (MMP) of the two mutant groups was lower than that of the wild group (P<0.01). Conclusion The mitochondria of HARS2 c.349G>A and c.908T>C mutant cells had certain respiratory defects and reduced proliferation ability, which may be the cytological mechanism of Perrault syndrome (PRLTS) induced by HARS2 mutation.