Identification of a Functional Genetic Variant at 16q12.1 for Breast Cancer Risk: Results from the Asia Breast Cancer Consortium
Figure 3
In vitro functional characterization of SNP rs4784227.
(A) Effect of rs4784227 on luciferase reporter activity. HEK 293, MCF10A, MCF-7, and MDA-231 cells were transiently transfected with pGL3 promoter vector and the constructs carrying the reference allele (C) and risk allele (T) of rs4784227, respectively. Relative luciferase activities are shown as mean ± SD of four experiments (relative to empty vector). Statistical analysis was done using Student's t-test comparing C and T alleles (* P<0.05, ** P<0.01, n = 4). (B) Electrophoretic mobility shift assays. Nuclear protein extracts from MCF-7 (top panel), MCF10A (middle panel), and MDA-231 (bottom panel) cells were incubated with biotin-labeled probes corresponding to reference allele C (lanes 1–5) or the risk allele T (lanes 6–10) of rs4784227 in the absence or presence of competitors. Lanes 1 and 6, no nuclear extracts; lanes 2 and 7, unlabeled competitor in 200-fold molar excess; lanes 3 and 8 (5 mM MgCl2), lanes 4 and 9 (2.5 mM MgCl2), and lanes 5 and 10 (1.25 mM MgCl2), no competitor. I: nonspecific DNA-protein complex bands from MCF-7, MCF10A, and MDA-231 cells; II: specific DNA-protein complex bands; III: free biotin-labeled probes.