| Background:Hepatocellular carcinoma is one of the most common malignant tumors worldwide. At the very early hepatocellular carcinoma usually has little or no symptoms until the clinical diagnosis, then most have occurred intrahepatic or extrahepatic metastasis which due to the main cause of the death in hepatocellular carcinoma patients. Tumor metastasis contains several steps:EMT, loss of cellular adhesion, increased motility and invasiveness, entry and survival in the circulation, exit into new tissue, and eventual colonization of a distant site. Lots of genes, transcription factors and cytokines were involved in, and there is interaction between them; this increases the difficulty to screening target genes or molecules for treatment. Recently, Han report that SATB1 play important role in breast cancer. Unlike the classic transcription factors which only bind special binding sites in individual target gene, SATB1 is a DNA-binding protein, locates in the vicinity of gene regulatory elements and regulates gene expression via binding the MAR sites on chromosomes. Here we detected SATB1 expression in tumor samples and hepatoma cells, and investigated the role of SATB1 in metastasis and invasion of HCC.Methods:Forty-five Chinese patients who underwent resection of hepatocellular carcinoma in thedepartment of Surgery, Tongji Hospital, Huazhong University of Science and Technology between 2008～2009. Cell lines used in the study include HCCLM8, MHCC-97H,SK-HEP-1,MHCC-97L,HepG2,SMMC-7721,Huh7,Hep3B. SATB1 was detected by Semi-quantitative PCR, western blotting, immunohistochemistry immunofluorescence in cells and HCC tissues, the relationship between SATB1 mRNA expressions with clinicopathological features was further analyzed by real-time PCR; SATB1 expression was inhibited by shRNA in SK-Hep-1 cell and over-expressed by transfected with the whole length of SATB1 cDNA in SMMC-7721 cell, expression microarray analysis was applied to find genes related to metastasis which were regulated by SATB1. Real-Time PCR and CHIP was used to verified the genes regulated by SATB1 in hepatocellular carcinoma, cell morphology was observed using inverted microscope, the cytoskeleton was observed using confocal laser scanning microscopy after F-actin staining by TRITC-labeled phalloidin; the cell ability of migration and invasion was examined by transwell analysis when downregulation/upregulation of SATB1 expression; tumor growth and metastasis assay was used to tested after downregulation/upregulation of SATB1 in HCC.Results:We detected SATB1 expression in 45 cases of HCC tissues and their corresponding non-tumorous liver samples,28 (62%) cases of the tumors and 18 (40%) cases of the non-tumorous liver samples were positive for SATB1, the SATB1 protein was located in nucleus, the mRNA and the protein of the SATB1 expression showed a higher level in tumor tissues than the non-tumorous liver samples. The relationship between SATB1 mRNA with clinicopathological features was further analyzed. The data suggested that SATB1 expression increased with the liver cirrhosis, tumor diameter, portal invasion, lymph node metastases and poor differentiation (p<0.05), it should be noted that a significant difference in the levels of SATB1 expression was observed between the Serum HBsAg positive and negative tissues (p< 0.05). Then we examined SATB1 expression in 8 kinds of hepatoma cell lines with different metastatic potential, including high metastatic potential cancer cell linesSK-HEP-1, HCCLM8, MHCC-97H (Ct value were 2.83±0.6,2.72±0.54,2.09±0.28), medium and low metastatic potential cell lines MHCC-97L, HepG2, SMMC-7721, Huh7 (Ct value were 0.29±0.1, 0.35±0.11,0.19±0.08,0.14±0.02), non metastatic potential cell lines Hep3B had little SATB1 expression. we knocked down SATB1 expression via SATB1-shRNA mediated RNA interference in human hepatoma SK-HEP-1 cell line; over-expressed SATB1 in SMMC-7721 by transfecting with the whole length of SATB1 plasmids pEGFP1-SATB1, Expression microarray analysis indicated that 1450 SATB1-activated genes and 1634 SATB1-repressed genes are marked by double-headed arrows between the shRNA-control and shRNA-SATB1 in SK-Hep-1 cells, while 1286 SATB1-activated genes and 1463 SATB1-repressed genes are marked by double-headed arrows between the pEGFP1-control and pEGFP1-SATB1 in SMMC-7721 cells. SATB1 regulated nearly 200 genes compared of the two sets of results which involved in invasion and metastasis of HCC, such as CDH3, FN1 adhesion molecules; TCF4 signal transduction genes; APC tumor suppressor gene; MMP12, CTSL1 protease; Etsl transcription factor; SLPI,SERPINB5,Vil2 gene. These genes were verified by Real-Time and CHIP and the results show that SATB1 regulated genes expression by binding the ATC sites in DNA. Down-regulation of SATB1 result in cell displaying cobblestone epithelial morphology instead of the usual typical long spindle shape using reverse microscope which showed in control group. Up-regulation of SATB1 leaded to cell elongating and lacking adherens junctions, the cell appeared an Epithelial-Mesenchymal Transition like changes. We also investigated the distribution of F-actin between the cells stably expressing SATB1 shRNA, full-length SATB1 cDNA and their corresponding control group. Down-regulation of SATB1 in SK-Hep-1 caused cytoskeleton disappeared and the microfilament beam scattered around the cells. Up-regulation of SATB1 induced filaments into stress fibers which were orientated parallel to the long axis of the cell and were present throughout the nucleoplasm. We detected vimentin, E-cadherin, tight junction protein 1 expression, and found knocking down of SATB1 expression in SK-HEP-1, the cell had increased expressions of E-cadherin(0.82±0.04 vs 0.42±0.01, p<0.05) and tight junction protein (0.62±0.02 vs 0.06±0.01, p<0.01) and a decreased expressions of vimentin(0.27±0.02 vs 0.57±0.06, p<0.05), while the opposite results appeared in the SMMC-7721 transfecxted with the pEGFPl-SATBl plasmids, the cell had decreased expressions of E-cadherin(0.36±0.02 vs 0.64±0.06, p<0.01) and tight junction protein (0.35±0.01 vs 0.51±0.04,p<0.05) and increased expressions of vimentin(0.64±0.03 vs 0.34±0.04, p<0.05). Then we examined whether downregulation/upregulation of SATB1 expression had effect on the cell migration and invasion. We evaluated the serum induced migration of cells using transwell migration and invasion assay. The results suggested that down-regulation of SATB1 resulted in significantly reduced ability in cell migration and invasion (the OD value was 0.16±0.02 vs 0.33±0.05, P< 0.01,0.76±0.12,0.22±0.06,p< 0.05), while up-regulation of SATB1 increased ability in migration and invasion (the OD value was 0.79±0.11 vs 0.26±0.04, P< 0.05,0.67±0.08 vs 0.26±0.04, p< 0.05). In order to observe the effect of SATB1 expression on the growth and migration of tumors in vivo, we established four xenograft tumor models in BALB/c nude mice. The mean weight of tumors of SATB1-shRNA of SK-Hep-1 was significantly lower than that of control group (experimental vs control 0.14±0.02g vs 0.43±0.06g, p<0.5). In contrast with the results of the above, the mean weight of tumors of pEGFP1-SATB1 of SMMC-7721 group was significantly more than those of control tumors groups (experimental vs control 0.57±0.05g vs 0.2±0.07g, p<0.5). As the BALB/c nude mice died during the observation, the result of tumor migration assay in vivo was not obtained.Conclusions:SATB1 expression was detected in HCC and cell lines, SATB1 expression was related to distant metastases, the SATB1 expression increased in high metastatic potential cells; down-expressed and over-expressed SATB1 could regulated many genes expression by binding the MARsites of gene DNA sequence; SATB1 induced cell changes in MET-EMT like morphology and cytoskeletal reorganization, and regulated Vimentin,Tight junction protein 1,E-cadherin expression, and result in decreased or increased ability in migration and invasion in vitro and tumor growth in vivo. |
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