Affiliation:
1. Tongji University School of Medicine
2. Shanghai Tenth People's Hospital
3. Shanghai Sunshine Rehabilitation Center
4. new york institute of technology college of osteopathic medicine at
5. Ningbo No. 2 Hospital
Abstract
Abstract
Background
Lung adenocarcinoma (LUAD) is the most common histological subtype of lung cancer, which is a serious threat to human health. Adenylate cyclase associated protein 1 (CAP1) is an important functional protein, which is closely related to the occurrence and progression of cancer.
Methods
In this study, we used the CRISPR-dCas9-Dnmt3a system to target the CAP1 promoter to construct LUAD cell strains that can steadily up-regulate the methylation of CAP1 promoter. The methylation specific PCR and Massarray methylation sequencing were used to detect the methylation of CAP1 promoter. The western blot and immunohistochemistry were used to detect protein expression. The functional changes of LUAD cells were detected by CCK-8 assay, colony formation assay, flow cytometry assay, wound healing assay and trans-well assay.
Results
In this study, we found that the CAP1 promoter was abnormally hypo-methylated in LUAD cells and tissues. The expression of CAP1 protein was higher in cancerous tissues compared to para-carcinoma tissues in early stage LUAD, and higher in A549, H1299 and PC9 cells than in Beas-2B control cells (P < 0.05). Up-regulating methylation of CAP1 promoter can reduce the expression of CAP1 protein, promote apoptosis of LUAD cells through Bax/Bcl-2/Caspase-3 pathway, and inhibit the migration and invasion of LUAD cells by acting together with Actin and Cofilin. The methylation of CAP1 promoter is regulated by Dnmt3a, Tet1 and/or Tet2.
Conclusions
These results suggest that abnormal hypo-methylation of CAP1 gene enhances biological characteristics of LUAD cells and up-regulating methylation of CAP1 promoter may be a potential treatment for LUAD.
Publisher
Research Square Platform LLC
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