Lv Wan-Qi, Wang Hai-Cheng, Peng Jing, Wang Yi-Xiang, Jiang Jiu-Hui, Li Cui-Ying
Central Laboratory, Peking University School and Hospital of Stomatology, Beijing 100081, China.
Department of Pathology, School & Hospital of Stomatology, Tongji University, Shanghai Engineering Research Center of Tooth Restoration and Regeneration, Shanghai 200072, China.
Oncotarget. 2017 Sep 21;8(62):105020-105036. doi: 10.18632/oncotarget.21136. eCollection 2017 Dec 1.
The low efficiency of clustered, regularly interspaced, palindromic repeats-associated Cas (CRISPR/Cas) system editing genes limits the application. A components of the extracellular matrix (ECM), the extra domain A positive fibronectin (EDA+FN), may be a target for CRISPR/Cas system for the pro-oncogenic effects. The exclusion of EDA exon would alter the microenvironment and inhibit tumor progression, even the frequency of gene editing is still limited.
The pro-oncogenic effects were confirmed by the exclusion of EDA exon from the fibronectin gene, as illustrated by the down-regulated proliferation, migration and invasion of CNE-2Z or SW480 cells (P<0.05). Furthermore, although the efficacy of EDA exon knockout through CRISPR/Cas system was shown to be low , the EDA+FN protein levels decrease obviously, inhibiting the tumor growth rate significantly (P<0.05), which was accompanied by a decrease in Ki-67 expression and microvessel numbers, and increased E-cadherin or decreased Vimentin expression (P<0.05).
Human nasopharyngeal carcinoma cell line CNE-2Z, and the colorectal carcinoma cell line SW480 were transfected with CRISPR/Cas9 plasmids targeting EDA exon. The effects of the exclusion of EDA on the cell proliferation, motility and epithelial-mesenchymal transition (EMT) were investigated, and the western blot and real-time PCR were performed to analyze the underlying mechanisms. Furthermore, CRISPR/Cas9 plasmids were injected into xenograft tumors to knockout EDA exon , and tumor growth, cell proliferation, EMT rate, or vascularization were investigated using western blot, PCR and immunohistochemistry.
CRISPR/Cas system targeting ECM components was shown to be an effective method for the inhibition of tumor progression, as these paracrine or autocrine molecules are necessary for various tumor cells. This may represent a novel strategy for overcoming the drug evasion or resistance, in addition, circumventing the low efficiency of CRISPR/Cas system .
成簇规律间隔短回文重复序列相关Cas（CRISPR/Cas）系统编辑基因的效率低下限制了其应用。细胞外基质（ECM）的一个成分，额外结构域A阳性纤连蛋白（EDA+FN），可能因其促癌作用而成为CRISPR/Cas系统的一个靶点。排除EDA外显子会改变微环境并抑制肿瘤进展，即便基因编辑的频率仍然有限。
从纤连蛋白基因中排除EDA外显子证实了其促癌作用，如CNE-2Z或SW480细胞的增殖、迁移和侵袭下调所示（P<0.05）。此外，尽管通过CRISPR/Cas系统敲除EDA外显子的效率较低，但EDA+FN蛋白水平明显降低，显著抑制肿瘤生长速率（P<0.05），这伴随着Ki-67表达和微血管数量的减少，以及E-钙黏蛋白表达增加或波形蛋白表达减少（P<0.05）。
用靶向EDA外显子的CRISPR/Cas9质粒转染人鼻咽癌细胞系CNE-2Z和结肠癌细胞系SW480。研究排除EDA对细胞增殖、运动性和上皮-间质转化（EMT）的影响，并进行蛋白质印迹和实时PCR分析潜在机制。此外，将CRISPR/Cas9质粒注射到异种移植瘤中以敲除EDA外显子，并使用蛋白质印迹、PCR和免疫组织化学研究肿瘤生长、细胞增殖、EMT率或血管生成。
靶向ECM成分的CRISPR/Cas系统被证明是抑制肿瘤进展的有效方法，因为这些旁分泌或自分泌分子对各种肿瘤细胞来说是必需的。这可能代表了一种克服药物逃避或耐药性的新策略，此外，还能规避CRISPR/Cas系统的低效率问题。
