线粒体tRNA5'剪切缺陷在母系遗传性高血压中的分子机制研究

Hypertension as a polygenic, multifactorial, and highly heterogeneous disorder could be caused by single-gene or multifactorial causes, resulting from interactions between environmental and inherited risk factors. In particular, mitochondria can regulate various aspects of vascular function, thereby being critical for the pathogenesis of hypertension. The maternal transmission of hypertension reported in several studies further supports mitochondrial involvement in hypertension. The human mitochondrial genome (mtDNA) encodes 13 subunits of the oxidative phosphorylation system, 2 rRNAs, and 22 tRNAs required for mitochondrial protein synthesis. Mitochondrial tRNA genes were proposed to be the hot spots for mutations associated with hypertension. These hypertension-associated tRNA mutations were the tRNA-Ile 4263A>G and 4295A>G mutations, the tRNA-Met 4435 A>G and 4454 T>C mutations, and the tRNA-Ala 5655 A>G. These mutations have structural and functional consequences, including the processing of RNA precursors, nucleotide modification, and aminoacylation As part of a genetic screening program for hypertension in a cohort of 2,070 Han Chinese hypertensive subjects, we identified the homoplasmic 4401A>G transition at the spacer immediately to the 5’end of tRNA-Met and tRNA-Gln genes in three genetically unrelated probands whose families exhibited a maternal transmission of hypertension. Moreover, we have observed in intro that the 4401 A>G mutation caused an 80% reduction in the efficiency of the 5’end processing of tRNA-Met precursor, and 70% in the efficiency of the 5-end processing of tRNA-Gln. To better understanding how the tRNA 5-end cleavage defect associate with the matrilineal hypertension, we have generated cybrids derived from affected and control subjects with human umbilical vein endothelial cells (HUVECs), seeking how the 5-end processing defect causing the mitochondrial dysfunction, and what happening in tRNA transcription and translation, and mitochondria respond followed. We hope our finding will provide new insights into the molecular mechanism, management, and treatment of matrilineal hypertension.

高血压是遗传和环境因素等多方面因素影响的复杂疾病。线粒体tRNA是导致母系遗传性高血压的分子基础之一，但分子机制十分复杂。前期通过对大量高血压样本的筛查,我们发现三个无亲缘关系的家系均携带m.4401A>G位点,突变位于线粒体重链tRNA-Met和轻链tRNA-Gln之间，前期体外实验证明了突变导致两个tRNA5’端剪切效率显著下降。本课题在此基础上，构建人脐静脉内皮细胞（HUVECs）为受体的转线粒体细胞系，在细胞水平拟1）明确tRNA5’端剪切缺陷影响HUVECs线粒体形态与功能；2)阐述5’端剪切缺陷调控线粒体基因转录和翻译的分子机制，3）探索HUVECs线粒体功能受损下的自噬应激。本项目的实施将有助于进一步诠释并丰富母系遗传性高血压的分子致病机制，同时为临床干预提供科学依据。

高血压是心血管疾病最主要的危险因素，会加重心脏病、脑卒中、肾衰竭、早死亡和残疾等疾病的负担。本项目前期在无亲缘关系的母系遗传的原发性高血压家系中发现了一个线粒体m.4401A>G 新突变。项目以脐带血内皮细胞（HUVEC）为核供体的转线粒体细胞系为细胞模型，从线粒体代谢、功能和形态及内皮细胞成血管等方面深入探讨高血压相关m.4401A>G 突变的分子致病机制。(1)揭示了 m.4401A>G 突变影响线粒体tRNA代谢水平，并下调整个轻链mRNA的转录和翻译。m.4401A>G 突变降低了tRNAMet 和tRNAGln 5’端RNaseP的剪切效率，进一步影响tRNAGln 所在的线粒体轻链其他tRNA和ND6 mRNA转录，即tRNA-Ala，tRNA-Asn，tRNA-Cys，tRNATyr，tRNASer（UCN），tRNAGlu，tRNAPro 和ND6的RNA 稳态水平分显著下降。突变细胞中的蛋白ND6仅为正常细胞的40%。(2)阐述了m.4401A>G直接影响血管内皮细胞线粒体功能和形态。m.4401A>G突变的细胞株线粒体膜电势下降了30%，线粒体ROS增高约31%，线粒体氧化磷酸化产生的ATP下降约30%。线粒体的呼吸链功能显著下降；m.4401A>G 突变的细胞株线粒体与对照组相比，形态变长，与线粒体分裂相关的动力蛋白Drp1的表达量下降了约65%，Pink/Parkin没有明显变化， LC3-I/(LC3-I+II)增加了约2.7倍，细胞自噬增强。(3)明确了线粒体功能障碍导致血管内皮细胞功能紊乱。携带m.4401A>G突变的HUVEC细胞生成血管和迁移能力显著下降，NO代谢显著下降。综上，线粒体突变剪切缺陷，进一步调控线粒体基因的转录和翻译，导致线粒体功能障碍，最终影响HUVECs细胞功能，丰富和拓展了母系遗传原发性高血压的致病机制和靶向线粒体功能的干预新策略。