The E2F3/miR-125a/DKK3 regulatory axis promotes the development and progression of gastric cancer

Abstract

Abstract Background Gastric cancer (GC) is one of the most common malignant tumours with high mortality and metastasis rates. E2F3, miR-125a and DKK3 have been reported to be involved in various cancer types, but their detailed roles in GC have not been fully understood. Methods A QRT-PCR assay was used to examine the expression of E2F3, miR-125a and DKK3 in metastatic and nonmetastatic GC tissues. DKK3 plasmids, DKK3 shRNA, miR-125a mimic and miR-125a inhibitor were transfected into BGC823 cells to evaluate the biological functions of DKK3 and miR-125a. A scratch wound healing assay and Transwell assay were utilized to determine the migratory and invasive ability of BGC823 cells transfected with the DKK3 plasmids, DKK3 shRNA, miR-125a mimic and miR-125a inhibitor. Moreover, qRT-PCR and WB analysis were used to analyse the mRNA and protein expression levels of metastasis-related genes after proper transfection. The target relationship between miR-125a and the DKK3 mRNA 3′UTR was determined by a dual luciferase reporter assay, while the interaction between E2F3 and miR-125a was analysed by a ChIP assay. Results The clinical data showed that the DKK3 expression level in metastatic GC samples was significantly less than that in nonmetastatic GC samples, whereas the E2F3 and miR-125a expression levels in metastatic GC samples were notably greater than those in nonmetastatic GC samples. Moreover, knockdown of DKK3 and overexpression of miR-125a markedly promoted the migratory and invasive abilities of GC cells. Additionally, the protein and mRNA expression levels of metastasis-related genes, including N-cadherin, Vimentin, MMP2 and MMP9, were markedly decreased in the DKK3 and miR-125a inhibitor groups compared to their control groups and markedly increased in the DKK3 shRNA and miR-125a groups compared with the control group. Finally, a dual luciferase reporter assay and ChIP assay showed that E2F3 binds to the miR-125a promoter and that the DKK3 mRNA 3′UTR is a direct target of miR-125a. Furthermore, analysis of Kaplan–Meier curves also confirmed the regulatory role of E2F3 on miR-125a. Additionally, BGC823 cells transfected with E2F3 plasmids and shRNA downregulated and upregulated the expression of DKK3, respectively. Conclusion Our results suggested that E2F3 might play a tumour-promoting role in the metastasis and progression of GC by regulating the miR-125a/DKK3 axis.

Keywords

• Gastric cancer (GC)
• E2F3/miR-125a/DKK3 regulatory axis
• Metastasis and progression of tumours