Figure 2

Analysis of TFPI-2 promoter. (A) Breast cancer cell lines were transfected with promoter-luciferase plasmid cloned from different cell lines, bearing respective genetic mutation. The promoter from different cell line has nearly the same luciferase activity in both MDA-MB-435 and MCF-7 cell lines. Results are shown as mean ± SD. The apparent lower level of promoter activity in MDA-MB-435 cell was not statistically significant (p = 0.342>0.05). (B) Luciferase reporter gene constructs with 5'ends between nucleotides -1436 and -144 and a common 3'end at +75 were transiently transfected into breast cancer cells. The minimal construct P-144 has the same luciferase activity as P-1436. (C) Linker-scan mutation analysis of TFPI-2 promoter -144 to +1 region. The fragment from -144 to the transcriptional start point (+1) of TFPI-2 promoter were systematically replaced with an BamH I & Xba I polylinker (GGATCCTCTAGA), the resulting sequence were analyzed for not introducing new transcription factor binding site by MatInspector [21]. SM1 indicate the promoter sequence from -12 to +1, and in turn every 12 bases from -144 to -12 was indicated by SM12 to SM2. Then the luciferase promoter reporter plasmids were transfected into MDA-MB-435 cell line and the reported luciferase activity was measured. Results are shown as mean ± SD relative to the wild type (*p < 0.05; **p < 0.001).