Mechanism of circular RNA homeodomain interacting protein kinase 3 in promotion of proliferation and migration of triple-negative breast cancer

Objective To investigate the mechanism of circular RNA homeodomain interacting protein kinase 3 (circHIPK3) in promotion of proliferation and migration of triple-negative breast cancer (TNBC) cells.

Methods Real-time quantitative polymerase chain reaction (qRT-PCR) was used to detect the expression levels of circHIPK3 in TNBC cell lines HCC1806, MDA-MB-231, HCC-70 and human normal breast cell line MCF-10A. The TNBC cell lines with high expression of circHIPK3 were divided into sh-NC group(transfected NC knockdown plasmid), sh-circHIPK3 group(transfected circHIPK3 knockdown plasmid), and the TNBC cell lines with low expression were divided into oe-NC group(transfected NC overexpressed plasmid) and oe-circHIPK3 group(transfected circHIPK3 overexpressed plasmid). The expression levels of circHIPK3 in cells of each group were detected by qRT-PCR; cell proliferation assay (MTS) was used to detect the cell proliferation activity of each group; transwell assay was used to detect the migrated ability of cells in each group; TOP/FOP flash luciferase reporter assay was used to detect the luciferase activity of cells in each group; the expressions of Wnt/β-catenin signaling pathway-related proteins Wnt3a, β-catenin, c-MYC and matrix metalloproteinase 2 (MMP-2) proteins in the cells of each group were detected by western blotting.

Results The expression level of circHIPK3 in TNBC cell lines was higher than that in human normal breast cell line MCF-10A, its expression was the highest in MDA-MB-231 cells, and the lowest expression in HCC-70 cells, the differences were statistically significant differences (P < 0.05). qRT-PCR results showed that the relative expression of circHIPK3 in the sh-circHIPK3 group was (0.36±0.06), which was lower than (0.98±0.03) in the sh-NC group, and the difference was statistically significant (t=16.008, P < 0.001). The relative expression of circHIPK3 in the oe-circHIPK3 group was (6.29±0.71), which was higher than (0.99±0.05) in the oe-NC group, the difference was statistically significant (t=12.897, P < 0.001). Compared with the sh-NC group, the proliferation activity and migration ability of MDA-MB-231 cells in the sh-circHIPK3 group were decreased, and compared with the oe-NC group, the proliferation and metastasis of HCC-70 cells in the oe-circHIPK3 group were higher (P < 0.05 or P < 0.01). Compared with sh-NC group, the luciferase activity of MDA-MB-231 cells in the sh-circHIPK3 group was decreased, the luciferase activity of HCC-70 cells in the oe-circHIPK3 group was increased compared with the oe-NC group (P < 0.001). The protein expressions of Wnt3a, β-catenin, c-MYC and MMP-2 in MDA-MB-231 cells in the sh-circHIPK3 group were lower than those in the sh-NC group, while were higher in the oe-circHIPK3 group than those in the oe-NC group (P < 0.05).

Conclusion CircHIPK3 can promote the proliferation and migration of TNBC cells by regulating Wnt/β-catenin signal pathway.