In MDA-MB-231 cells, The mRNA optical density ratio(ODR: MTA1/18SrRNA) of MTA1 in the blank control, negative CB-839 order control and test groups (pGM1, pGM2) were 0.8097 ± 0.0173, 0.8119 ± 0.0367, 0.3623 ± 0.0087 and 0.1742 ± 0.0094, respectively. The statistical analysis showed that MTA1 mRNAs of MDA-MB-231 cells in the pGM1 and pGM2 groups were down-regulated significantly after transfection with either plasmids pGM1 or pGM2, compared with that in the blank group(P < 0.05). The inhibition rates were 55.3% and 78.5% in the pGM1 and pGM2 AR-13324 group, respectively. In MCF-7 cells, ODR in pGM1 and pGM2 group were 0.2386 ± 0.0018

and 0.1455 ± 0.0075, respectively. Compared to blank control group (ODR:0.4236 ± 0.0069) and negative control(ODR:0.4148 ± 0.0058), there were statistical difference(P < 0.05). MTA1 mRNA inhibition

rate for pGM1 and pGM2 were 43.7%, 65.7%. Thus, MDA-MB-231/pGM2 and MCF-7/pGM2 cell clones were chosen for further experiments. (Figure 3) Figure 3 MTA1 specific shRNAs results in the reduction of MTA1 mRNA levels in MDA-MB-231 and MCF-7 cells. A: mRNA levels of MTA1 in JIB04 manufacturer MDA-MB-231. M:DNA Marker. lane 1:Blank control group. lane 2: PG group(empty vector). lane 3: PGM1 group(the first pair pGenesil-1/MTA1-shRNA). lane 4:PGM2 group(the second pair pGenesil-1/MTA1-shRNA). B: mRNA levels of MTA1 in MCF-7. M:DNA Marker. lane 1:Blank control group. lane 2: PG group(empty vector). lane 3:PGM1 group(the first pair pGenesil-1/MTA1-shRNA). lane 4:PGM2 group(the PIK3C2G second pair pGenesil-1/MTA1-shRNA). C: Column diagram analysis for mRNA levels of MTA1, MTA1 specific shRNAs resulted in the reduction of MTA1 mRNA levels in MDA-MB-231 and MCF-7 cells (*P < 0.05). Influence of pGenesil-1/MTA1 shRNA vectors on ER alpha, MMP-9 and CyclinD1 protein expression in MDA-MB-231 and MCF-7 cells by Western blot analysis Results in two breast cancer cells by Western blot ananlysis indicated that, ER alpha was recovered positive in ER-negative human breast cancer cell lines MDA-MB-231, and protein levels of MMP-9 and CyclinD1 were down-regulation (P < 0.05). However, in ER alpha-positive

breast cancer cells MCF-7, protein expression levels of ER alpha, MMP-9 and CyclinD1 had no distinct difference in three groups(P > 0.05). (Figure 4) Figure 4 Western blot analysis for ER alpha, CyclinD1 and MMP-9 in MDA-MB-231 and MCF-7 cells. A: Western blot analysis for ER alpha, CyclinD1 and MMP-9. lane 1: blank control group in MDA-MB-231 cells. lane 2: PG group (empty vector) in MDA-MB-231 cells. lane 3:PGM2 group (the second pair pGenesil-1/MTA1 shRNA plasmid) in MDA-MB-231 cells. lane 4: blank control group in MCF-7 cells. lane 5: PG group(empty vector) in MCF-7 cells. lane 6:PGM2 group in MCF-7 cells. B: Column diagram analysis for protein expression of ER alpha, cyclinD1, MMP-9 in MDA-MB-231 and MCF-7 cells by Western blotting.1-3: blank control group, PG group and PGM2 group in MDA-MB-231 cells, respectively.