Fig. 4

LncRNA NCALD can induce the colony formation of luminal BC cells by regulating the expression of GRHL2. The lncRNA NCALD positively correlated with GRHL2 based on TCGA database data (panel a). Knockdown of lncRNA NCALD resulted in the downregulation of GRHL2 expression at the RNA level, (n = 3, mean ± SD; ***P < 0.001) (panel b). The level of GRHL2 significantly decreased in cells after lncRNA NCALD knockdown. The level of GRHL2 in cells transfected with GRHL2 overexpressing plasmid and lncRNA NCALD normal control plasmid (GRHL2 OV-lncRNA NC) was significantly higher compared to cells transfected with lncRNA NCALD control plasmid and GRHL2 normal control plasmid (GRHL2 NC-lncRNA NC). The levels of GRHL2 in cells transfected with GRHL2 normal control plasmid and lncRNA NCALD knockdown plasmid (GRHL2 NC-sh lncRNA) were significantly lower compared to those in the GRHL2 NC-lncRNA NC cells. Cells transfected with lncRNA NCALD knockdown plasmid and GRHL2 overexpressing plasmid can restore the depleted level of GRHL2 caused by lncRNA NCALD knockdown (panel c). All experiments were biologically replicated three times. Cell proliferation induced by knockdown of lncRNA NCALD was rescued by overexpression of GRHL2 in the colony formation assay (***P < 0.001) (panel d). All experiments were biologically replicated three times. The expression of cell proliferation-related proteins cyclin D and cyclin E were reduced, while the expression of p16 and p21 were increased in MCF7 or T47D cell line with knockdown of lncRNA NCALD. Furthermore, the restoration of GRHL2 led to a significant reversal of these expression changes (panel e). All experiments were biologically replicated three times