Luo Yue-Bei, Mitrpant Chalermchai, Adams Abbie M, Johnsen Russell D, Fletcher Sue, Mastaglia Frank L, Wilton Steve D
Centre for Neuromuscular and Neurological Disorders, Australian Neuro-Muscular Research Institute, University of Western Australia, Perth, Australia; Department of Neurology, Xiangya Hospital, Central South University, Changsha, China.
Centre for Neuromuscular and Neurological Disorders, Australian Neuro-Muscular Research Institute, University of Western Australia, Perth, Australia; Department of Biochemistry, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, Thailand.
PLoS One. 2014 Jun 3;9(6):e98306. doi: 10.1371/journal.pone.0098306. eCollection 2014.
We sought to use splice-switching antisense oligonucleotides to produce a model of accelerated ageing by enhancing expression of progerin, translated from a mis-spliced lamin A gene (LMNA) transcript in human myogenic cells. The progerin transcript (LMNA Δ150) lacks the last 150 bases of exon 11, and is translated into a truncated protein associated with the severe premature ageing disease, Hutchinson-Gilford progeria syndrome (HGPS). HGPS arises from de novo mutations that activate a cryptic splice site in exon 11 of LMNA and result in progerin accumulation in tissues of mesodermal origin. Progerin has also been proposed to play a role in the 'natural' ageing process in tissues. We sought to test this hypothesis by producing a model of accelerated muscle ageing in human myogenic cells. A panel of splice-switching antisense oligonucleotides were designed to anneal across exon 11 of the LMNA pre-mRNA, and these compounds were transfected into primary human myogenic cells. RT-PCR showed that the majority of oligonucleotides were able to modify LMNA transcript processing. Oligonucleotides that annealed within the 150 base region of exon 11 that is missing in the progerin transcript, as well as those that targeted the normal exon 11 donor site induced the LMNA Δ150 transcript, but most oligonucleotides also generated variable levels of LMNA transcript missing the entire exon 11. Upon evaluation of different oligomer chemistries, the morpholino phosphorodiamidate oligonucleotides were found to be more efficient than the equivalent sequences prepared as oligonucleotides with 2'-O-methyl modified bases on a phosphorothioate backbone. The morpholino oligonucleotides induced nuclear localised progerin, demonstrated by immunostaining, and morphological nuclear changes typical of HGPS cells. We show that it is possible to induce progerin expression in myogenic cells using splice-switching oligonucleotides to redirect splicing of LMNA. This may offer a model to investigate the role of progerin in premature muscle ageing.
我们试图通过增强早老素的表达来建立一个加速衰老的模型，早老素由人类成肌细胞中错误剪接的核纤层蛋白A基因（LMNA）转录本翻译而来。早老素转录本（LMNA Δ150）缺少外显子11的最后150个碱基，并被翻译成一种截短的蛋白质，与严重的早老性疾病——哈钦森-吉尔福德早衰综合征（HGPS）相关。HGPS源于从头突变，这些突变激活了LMNA外显子11中的一个隐蔽剪接位点，导致早老素在中胚层来源的组织中积累。早老素也被认为在组织的“自然”衰老过程中起作用。我们试图通过在人类成肌细胞中建立加速肌肉衰老的模型来验证这一假设。设计了一组剪接转换反义寡核苷酸，使其与LMNA前体mRNA的外显子11退火，并将这些化合物转染到原代人类成肌细胞中。逆转录-聚合酶链反应（RT-PCR）表明，大多数寡核苷酸能够改变LMNA转录本的加工过程。在早老素转录本中缺失的外显子11的150个碱基区域内退火的寡核苷酸，以及靶向正常外显子11供体位点的寡核苷酸，均可诱导产生LMNA Δ150转录本，但大多数寡核苷酸也会产生不同水平的缺失整个外显子11的LMNA转录本。在评估不同的寡聚物化学性质时，发现吗啉代磷酰胺二胺寡核苷酸比在硫代磷酸酯主链上带有2'-O-甲基修饰碱基的等效寡核苷酸序列更有效。吗啉代寡核苷酸诱导了核定位的早老素，免疫染色证明了这一点，以及HGPS细胞典型的形态学核变化。我们表明，使用剪接转换寡核苷酸来重定向LMNA的剪接，有可能在成肌细胞中诱导早老素的表达。这可能为研究早老素在肌肉过早衰老中的作用提供一个模型。
