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

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.

The extra domain A of fibronectin facilitates osteoclastogenesis in radicular cysts through vascular endothelial growth factor

“…To illustrate the function of the EDA fragment, the EDA exon was knocked out from the genome with the CRISPR/Cas system. Consistent with previous studies (Wang et al 2015a, Wang et al 2015b, Lv et al ) on EDA exon knockout, protein levels of EDA + FN were significantly decreased, but total FN was virtually unchanged. Consequently, conditioned medium from the EDA knockout group developed significantly decreased Trap + MNCs ( P < 0.05) similar to IST‐9 blockage, suggesting that osteoclastogenic induction of EDA + FN could be attributed primarily to function of the EDA fragment, rather than the entire protein.…”

“…As described in previous studies (Wang et al 2015a, Wang et al 2015b, Lv et al ), EDA exon was knocked out from the genome using the CRISPR/Cas system in order to elucidate its role in osteoclastogenesis. As illustrated by the PCR products, EDA knockout fibroblasts generated both the bands containing (675 bp) and without the EDA exon (415 bp; Fig.…”

“…Using the same protocol as in a previous studies (Wang et al 2015a, Wang et al 2015b, Lv et al ), at 70%–90% confluence, cells were cultured in serum‐free medium for 6 h, and plasmids containing sequences of single guide (sg) RNAs that complement the DNA flanking EDA were co‐transfected, using Lipofectamine 2000, according to manufacturer's protocol (Life Technologies, Carlsbad, CA, USA). Primer sequences and sgRNAs have been described in previous studies (Wang et al 2015a, Wang et al 2015b, Lv et al , Table ). These fibroblasts were the EDA knockout group.…”

“…Fibronectin (FN) is the main component of the ECM, and in most normal adult tissues, isoforms containing domains EDA (EDA + FN), EDB (EDB + FN) and IIICS (CS1‐FN) are absent (Liu et al , Lv et al ). However, myofibroblasts generate these isoforms via alternative splicing of the FN gene, especially during inflammation, wound healing or tumour formation (Kalluri & Zeisberg ), suggesting their involvement in bone destruction (Elmusrati et al ).…”

“…The effects of FN isoforms on osteoclastogenesis may contribute to bone destruction due to the inflammatory microenvironment of radicular cysts (Khan et al , Khan et al , Liu et al ). Since the three main splice variants, EDA + FN, EDB + FN and CS1‐FN, are ligands of integrin receptors and Toll‐like receptor 4 (TLR4), interactions between FN isoforms and receptors in pre‐osteoclasts may directly affect osteoclastogenesis (Doddapattar et al , Jiang et al ), whilst their interaction in other cells could alter the expression of osteoclastogenic genes, such as vascular endothelial growth factor (VEGF) or IL‐17 (Liu et al , Lv et al ), by which osteoclastogenesis can be indirectly influenced. Therefore, this study explored the effects of FN isoforms on bone destruction in radicular cysts, as well as the underlying mechanism through antibody blocking and gene editing using the type II bacterial clustered, regularly interspaced, palindromic repeats (CRISPR)‐associated (Cas) system (Kohan et al , Wang et al 2015a, Wang et al 2015b).…”