Figure 4

miR-130b-5p directly silences CCNG2 expression in triple-negative breast cancer cells. (A) Schematic diagram of the putative miR-130b-5p binding site in the 3′-UTR of CCNG2. The possible binding sequence of miR-130b-5p with the 3′-UTR of CCNG2 is shown in red. (B) Luciferase assays of CCNG2 in miR-130b-5p-overexpressed HEK-293 T cells. Two luciferase reporter constructs, Luc-CCNG2-UTR and Luc-CCNG2-UTR-mt3, were made to test whether miR-130b-5p directly targets the putative binding site in the 3′-UTR of CCNG2. The mutated nucleotides are indicated in red. Compared with the negative control, overexpression of miR-130b-5p was shown to significantly inhibit luciferase reporter activity with Luc-CCNG2-UTR, but not with Luc-CCNG2-UTR-mt3. Each bar represents the mean of triplicate measurements ± the SD; *p <0.05. (C) Endogenous expression analysis of CCNG2 in MDA-MB-231 cells transfected with lentiviral vectors containing miR-130b-5p precursor sequences. The endogenous expression levels of CCNG2 were significantly repressed after the forced expression of miR-130b-5p in MDA-MB-231 cells. (D) Comparisons of miR-130b-5p expression in normal breast tissues (n = 14), early-stage triple-negative breast cancers (n = 7), and advanced-stage triple-negative breast cancers (n = 17). MiRNA expression reads were normalized and analyzed in log₁₀ scale. miR-130b-5p expression was significantly associated with both early-stage and advanced-stage triple-negative breast cancers.