Wang et al. Journal of Experimental & Clinical Cancer Research 2010, 29:140 http://www.jeccr.com/content/29/1/140 Page 4 of 7 Table 1 Clinical and pathological characteristics of patients Table 2 Expression of HBO1 in relation to the clinical and pathological characteristics of patients characteristic Tumor grade (n[%]) Ki67 status (n[%]) Estrogen receptor a status (n[%]) Progesterone receptor status (n[%]) I II/III negative positive negative positive negative positive Negative Positive negative positive G1 G2/G3 P53 status (n[%]) HBO1 status (n[%]) Histology Grade (n[%]) value 45 [40.2%] 67 [59.8%] 50 [44.6%] 62 [55.4%] 39 [34.8%] 73 [65.2%] 60 [53.6%] 52 [46.4%] 62 [55.4%] 50 [44.6%] 48 [42.9%] 64 [57.1%] 32 [28.6%] 80 [71.4%] 17b-estradiol (E2). Quantitative real-time PCR was used to determine the effect of E2 on the mRNA level of HBO1. With the dose of E2 increasing from 10-9 to 10-7 M, the mRNA level of HBO1 gradually increased and reached a plateau at 10-8 M in T47 D cells (Figure 2A). Hereby, 10-8 M of E2 was applied for all subsequent experiments. Western blot was also performed to con- firm that E2 could upregulate HBO1 protein expression in T47 D (Figure 2B) and MCF-7 cells (Figure 2C). Taken together, these results demonstrated that E2 increased expression of HBO1 at transcriptional level. E2-induced HBO1 expression is inhibited by ICI 182,780 or ERa RNAi To further study the role of ERa in HBO1 expression, ICI 182,780 was further applied in T47 D cells (Figure 3A) and MCF-7 cells (Figure 3B) to block the effect of E2. As shown in Figure 3A, E2 increased HBO1 protein expression (lane 2), which was significantly reduced by ICI 182,780 (lane 4). Similar results were obtained in MCF-7 cells (Figure 3B). ICI 182,780 was reported to act by binding ERa, causing disassociation of ERa asso- ciated proteins and resulting in impaired receptor dimerisation and increased receptor degradation [12,13]. As expected, ERa expression was decreased by ICI 182,780 (Figure 3A and 3B). These results indicated that clinical feature total Tumor grade HBO1 expression negative positive I II/III Ki67 negative positive negative positive negative positive P53 negative positive G1 G2/G3 G1 G2/G3 G1 G2/G3 Estrogen receptor Progesterone receptor Histology Grade among ER positive tumors Histology Grade among ER negative tumors Histology Grade *P < 0.05 **P < 0.01 45 67 50 62 39 73 60 52 62 50 32 80 25 48 8 31 21 28 23 25 26 22 34 14 23 25 17 31 12 10 5 21 24 39 27 37 13 51 26 38 39 25 15 49 13 38 3 10 P-value 0.610 0.550 < 0.001** 0.002** 0.170 0.165 0.016* 0.779 E2-induced HBO1 upregulation could be inhibited by ICI 182,780. In order to figure out the role of ERa in E2-induced HBO1 upregulation, ERa siRNA was trans- fected into T47 D and MCF7 cells to knockdown ERa. We observed that E2-induced HBO1 upregulation was inhibited by ERa siRNA (lane 4), indicating that HBO1 might be a downstream signaling molecule for ERa. ERK1/2 signaling pathway was involved in the expression of HBO1 increased by E2 Using TRSEARCH software based on the sequence data- base, the promoter region of the HBO1 gene has no putative binding sites for ERa (data not shown). And previous studies have shown that steroidal estrogens are able to rapidly and transiently activate the ERK1/2 path- way [13], so we examined whether the ERK1/2 pathway was involved in the effect of E2 on HBO1 expression in breast cancer cells. As shown in Figure 4, E2-increased HBO1 protein expression was significantly suppressed