title: The CRISPR/Cas system inhibited the pro-oncogenic effects of alternatively spliced fibronectin extra domain A via editing the genome in salivary adenoid cystic carcinoma cells.
abstract: OBJECTIVE
  To identify the association of fibronectin (FN) extra domain A (EDA) with the progression of salivary adenoid cystic carcinoma (SACC). Accordingly, the exclusion of EDA exon through the CRISPR/Cas9 system was investigated as the rescue for such pro-oncogenic splicing.
  
  
  METHODS
  SACC-83 cells were transiently transfected with plasmids containing recombinant EDA, and the cellular growth and motility were then accessed in vitro. Epithelial-mesenchymal transition (EMT) was investigated with immunohistochemistry, Western blot, and real-time PCR analysis. SACC tissues from 81 patients were used to access the associations between EDA+FN and clinical-pathological parameters. CRISPR/Cas9 plasmids containing sgRNA were designed and co-transfected into SACC-83 cells; the effects of EDA knockout on cellular growth and motility were then accessed.
  
  
  RESULTS
  The recombinant EDA exhibited little effect on the proliferation of SACC cells, but significantly promoted the migration and invasion of the cells (P < 0.05), accompanied with upregulated EMT (P < 0.05); consistently, the expression of EDA+FN was positively associated with the metastasis, nerve invasion and recurrence of SACC (P < 0.05). Furthermore, the EDA knockout from the FN gene in most SACC cells resulted in a decrease in cell motility and invasion, as well as prolonged population doubling time, compared with untreated SACC-83 cells (P < 0.05).
  
  
  CONCLUSIONS
  The EDA domain significantly promoted the motility of SACC cells, and positively associated with the tumor progression in patients with SACC. Thus, it is a potential risk factor and also a therapeutic target for SACC. The CRISPR/Cas9 system may control a pro-oncogenic splicing process through the exclusion of EDA exon from the FN gene, leading to inhibition of motility, invasion and proliferation of cancer cells.
authors: The Central Laboratory, Peking University School and Hospital of Stomatology, Haidian District, Beijing, China.
authors: H-C
authors: HC
authors: Wang
authors: The Central Laboratory, Peking University School and Hospital of Stomatology, Haidian District, Beijing, China.
authors: Y
authors: Y
authors: Yang
authors: Department of Oral Implanting, Shandong University School of Stomatology, Lixia District, Jinan, China.
authors: S-Y
authors: SY
authors: Xu
authors: The Central Laboratory, Peking University School and Hospital of Stomatology, Haidian District, Beijing, China.
authors: J
authors: J
authors: Peng
authors: The Department of Orthodontics, Peking University School and Hospital of Stomatology, Haidian District, China.
authors: J-H
authors: JH
authors: Jiang
authors: The Central Laboratory, Peking University School and Hospital of Stomatology, Haidian District, Beijing, China.
authors: C-Y
authors: CY
authors: Li
mesh_terms: D017398:Alternative Splicing; D064113:CRISPR-Cas Systems; D003528:Carcinoma, Adenoid Cystic / Q000235:genetics* / Q000378:metabolism / Q000473:pathology / Q000628:therapy; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000235:genetics; D058750:Epithelial-Mesenchymal Transition / Q000235:genetics; D005091:Exons; D005260:Female; D005353:Fibronectins / Q000096:biosynthesis* / Q000235:genetics*; D055786:Gene Knockout Techniques; D006801:Humans; D008297:Male; D008875:Middle Aged; D009361:Neoplasm Invasiveness; D009362:Neoplasm Metastasis; D009364:Neoplasm Recurrence, Local / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D012307:Risk Factors; D012468:Salivary Gland Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology / Q000628:therapy; D014162:Transfection; D015854:Up-Regulation
chemical_list: D005353:Fibronectins; C574442:extra domain A fibronectin, human
keywords: CRISPR/Cas; cellular motility; extra domain A; knockout; pro-oncogenic splicing; salivary adenoid cystic carcinoma