Calcium/calmodulin-dependent protein kinase II inhibitor I (CAMK2N1) is one of the tumor suppressor genes in prostate cancer (PCa) and is significantly downregulated in PCa tissues compared to benign and normal prostate tissues. Reduced expression of CAMK2N1 is positively correlated with PCa progression. However, the mechanism of CAMK2N1 silencing in PCa is still unclear. The promoter region of CAMK2N1 contained abundant CG loci, providing a great possibility for DNA methylation. Consequently, we postulated that epigenetic modification resulted in the abnormal expression of CAMK2N1 in PCa.
Firstly, we determined the DNA methylation level of CAMK2N1 in prostate cell lines and clinical specimens by bisulfite sequencing (BS), pyrosequencing and The Cancer Genome Atlas in silico analysis. Subsequently, we explored the expression of CAMK2N1 and its DNA methylation level by qRT-PCR, western blot, immunofluorescence, BS and methylation-specific PCR in PCa cells after 5-Aza-CdR treatment or DNMT1 gene modification. Moreover, we analyzed DNMT1 expression as well as the related signaling pathways in CAMK2N1 upregulated or downregulated PCa cells. Finally, functional assays including wound healing, invasion and migration assay, and xenograft model in nude mice were used to investigate the effect of DNMT1/CAMK2N1 interaction on the progression of PCa.
CAMK2N1 was highly methylated in PCa cells and tissues compared to normal prostate epithelial cells, normal prostate tissues and benign prostatic hyperplasia BPH tissues. The hypermethylation of CAMK2N1 was associated with the clinicopathological characteristics in PCa patients. The reduced expression of CAMK2N1 can be restored by 5-Aza-CdR treatment via demethylation. Moreover, we confirmed that DNMT1 formed a positive feedback loop with CAMK2N1 in PCa cells. CAMK2N1 expression was downregulated by DNMT1-mediated DNA methylation, which reversely induced DNMT1 expression through the AKT or ERK signaling pathway. The results of in vitro and in vivo experiments demonstrated that CAMK2N1 inhibited PCa cell invasion, migration and proliferation and these effects were reversed by DNMT1.
DNMT1-mediated hypermethylation of CAMK2N1 not only downregulates gene expression but also promotes the progression of PCa, which could be served as a potential predictive biomarker.