Long non-coding RNA NEAT1 promotes sarcoma metastasis by regulating RNA splicing pathways

To identify genes that are differentially expressed in lung metastases of sarcoma, we generated KrasLSL-G12D; Trp53Flox/Flox (KP) mice which develop autochthonous sarcomas in the muscle following injection of an adenovirus expressing Cre recombinase. Cre activates expression of mutant KrasG12D and deletes both alleles of Trp53. As each primary sarcoma reached the size of around 300 mm3, the leg bearing the tumor was amputated and the tumor stored for subsequent RNA sequencing. The amputated mice were followed for several months and 6 mice developed 14 lung metastases: two mice developed one lung metastases, two mice developed two lung metastases, and two mice developed four lung metastases respectively. RNA was then extracted from the 6 primary tumors and the 14 matched lung metastases. Therefore, a total of 20 RNA samples were sent for RNA sequencing Overall design: a total of 20 RNA from the 6 primary tumors and the 14 matched lung metastases were sent for RNA sequencing

为鉴定肉瘤肺转移灶中的差异表达基因，我们构建了KrasLSL-G12D; Trp53Flox/Flox（KP）小鼠模型：该品系小鼠在注射表达Cre重组酶（Cre recombinase）的腺病毒后，可在肌肉组织中自发形成肉瘤。Cre重组酶可激活突变型KrasG12D的表达，并敲除Trp53的两个等位基因。当单枚原发肉瘤体积达到约300 mm³时，摘除荷瘤下肢并将肿瘤保存，用于后续RNA测序。对术后小鼠进行长达数月的随访，最终6只小鼠共出现14处肺转移灶：其中2只小鼠各出现1处肺转移灶，2只各出现2处，剩余2只各出现4处。随后从6枚原发肿瘤及14枚配对肺转移灶中提取RNA，共计获得20份RNA样本，用于RNA测序。实验整体设计：将来自6枚原发肿瘤与14枚配对肺转移灶的共计20份RNA样本提交进行RNA测序。