Abstract
BackgroundThe 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.ResultsThe 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 materialsHuman 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.ConclusionCRISPR/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.
Highlights
The conventional treatment strategies attempting to eradicate tumor cells as radical as possible, are frequently inefficient, because of the evasion from drugs, where the drug-sensitive tumor cells are eliminated, but the pre-existing insensitive sub-clones are selected and are able to survive [1, 2]; and drug resistance development represents an evolution of cancer cells, through mutations or reprogramming metabolic patterns due to the unstable genomes [3, 4]
CRISPR/Cas system targeting extracellular matrix (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
Summary
The conventional treatment strategies attempting to eradicate tumor cells as radical as possible, are frequently inefficient, because of the evasion from drugs, where the drug-sensitive tumor cells are eliminated, but the pre-existing insensitive sub-clones are selected and are able to survive [1, 2]; and drug resistance development represents an evolution of cancer cells, through mutations or reprogramming metabolic patterns due to the unstable genomes [3, 4]. Paracrine or autocrine molecules participate in maintaining favorable conditions for the propagation of tumor cells, such as the extracellular matrix (ECM) component extra domain A positive fibronectin (EDA+FN), as well as the vascular endothelial growth factor A isoforms (VEGF-Axxx), the pro-oncogenic isoforms generated by the alternative splicing of FN or VEGF genes [9, 10], respectively. These alternative spliced isoforms are always absent in normal adult tissues, but exclusively expressed in tumor, wound healing and inflammation. The exclusion of EDA exon would alter the microenvironment and inhibit tumor progression, even the frequency of gene editing is still limited
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