In particular, the expression of c-Myb was at a high level in metastatic HCC cell line HCCLM6 and MHCC97-L SB525334 chemical structure cells, and at a much lower level in SMMC-7721 cells, and barely detectable in normal cell line L02 cells. Corresponding to different OPN expression level (HCCLM6 > MHCC-97-L> SMMC-7721),

the expression level of c-Myb increased sharply in HCCLM6 cells (Figure 1A). Similar results were obtained in real-time PCR analysis. When normalized to the internal standard control, mRNA expression of c-Myb in HCCLM6 cells was significantly higher than SMMC-7721 cells (Figure 1B). Similar to the result of mRNA expression, the difference of c-Myb protein expression between HCCLM6 and SMMC-7721 cells was also significant. (Figure 1C) Figure 1 Verification

of difference of OPN and c-Myb expression in HCC cell lines. HCCLM6 cells expressed high level of OPN and c-Myb compared with SMMC-7721 cells. (A) Relative OPN and c-Myb mRNA levels in different cell lines by RT-PCR analysis. (B) Real-time quantitative PCR analysis confirmed the difference of c-Myb mRNA expression in different cell lines. Graph depicted relative expression of OPN mRNA normalized to that of GAPDH. The mRNA expression of c-Myb in HCCLM6 was used as control. Data expressed as means ± SD (* P < 0.05, SMMC-7721 vs. HCCLM6). (C)Western blot analysis of OPN and c-Myb protein expression in HCC cell line SMMC-7721 and HCCLM6. Blot was representative of three experiments. Table 2 Differential this website activity of transcription factorsin two HCC cell lines (SMMC-7721, HCCLM6) with different OPN

expression levels (> 2 fold or <0.5-fold Rolziracetam change) Name HCCLM6/SMMC-7721 ratio Description Up-regulation     MAZ 3.10 MYC-associated zinc finger protein E4BP4 2.86 nuclear factor, IL- 3 regulated c-Myb 2.80 v-myb myeloblastosis viral oncogene GATA-2 2.74 GATA binding protein 2 TEF1 2.73 activator PEBP2 2.39 polyoma enhancer binding protein 2 Smad3/4 2.27 MADH3/4 IRF-1/2 2.21 interferon regulatory factor 1/2 PEBP 2.13 polyoma enhancer binding protein GAG 2.13 amyloid precursor protin (APP) regulator ADR1 2.10 alcohol dehydrogenase regulatory gene 1 Down-regulation     NF-E2 0.19 nuclear factor (erythroid-derived 2), 45 kDa EGR 0.21 early growth response C/EBPα 0.22 CCAAT/enhancer binding protein alpha E2F-1 0.28 E2F transcription factor 1 CYP1A1 0.30 cytochrome P450-c HiNF-A 0.31 A nuclear protein Sp1 0.31 Sp1 transcription factor E12/E47 0.31 enhancer binding factors E12/E47 PARP 0.34 poly(ADP-ribose) synthetase/polymerase ELK1 0.34 member of ETS oncogene family E4F1 0.34 E4F transcription factor 1 3.2 Transcription factor c-Myb contributing to transcription activation of the OPN promoter in HCCLM6 cells Having shown that c-Myb was over-expressed in HCCLM6 cells, we next sought to establish whether it has a functionally important role in the regulation of OPN expression.