Oncotarget

Research Papers:

Gene editing of the extra domain A positive fibronectin in various tumors, amplified the effects of CRISPR/Cas system on the inhibition of tumor progression

Wan-Qi Lv, Hai-Cheng Wang, Jing Peng, Yi-Xiang Wang, Jiu-Hui Jiang and Cui-Ying Li _

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Oncotarget. 2017; 8:105020-105036. https://doi.org/10.18632/oncotarget.21136

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Abstract

Wan-Qi Lv1,*, Hai-Cheng Wang2,*, Jing Peng3, Yi-Xiang Wang1, Jiu-Hui Jiang4 and Cui-Ying Li1

1Central Laboratory, Peking University School and Hospital of Stomatology, Beijing 100081, China

2Department of Pathology, School & Hospital of Stomatology, Tongji University, Shanghai Engineering Research Center of Tooth Restoration and Regeneration, Shanghai 200072, China

3Department of Beijing Citident Stomatology Hospital, Beijing 100032, China

4Department of Orthodontics, Peking University School and Hospital of Stomatology, Beijing 100081, China

*The authors have contributed equally to this article

Correspondence to:

Cui-Ying Li, email: licuiying_67@163.com

Jiu-Hui Jiang, email: drjiangw@gmail.com

Keywords: CRISPR/Cas, extracellular matrix, extra domain A fibronectin, gene editing, tumor progression

Received: December 28, 2016     Accepted: July 30, 2017     Published: September 21, 2017

ABSTRACT

Background: The low efficiency of clustered, regularly interspaced, palindromic repeats-associated Cas (CRISPR/Cas) system editing genes in vivo 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.

Results: 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 in vivo, 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).

Methods and materials: 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 in vivo, and tumor growth, cell proliferation, EMT rate, or vascularization were investigated using western blot, PCR and immunohistochemistry.

Conclusion: 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 in vivo.


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