【作者】 罗敏；

【导师】 田德安；

【作者基本信息】 华中科技大学 ， 内科学， 2010， 博士

【摘要】 目的原发性肝癌为最常见恶性肿瘤之一,肝癌患者临床确诊时大部分已出现肝内或肝外转移,是导致死亡的主要原因。肿瘤侵袭转移过程涉及细胞EMT改变、癌细胞迁移等多个方面,许多与肿瘤侵袭转移有关的基因、转录因子及细胞因子等参与其中,彻底阐明它们的相互作用极为困难。最近研究发现核基质结合蛋白SATB1在乳腺癌转移调控中起着重要作用。SATB1与传统转录因子识别特异基因特殊位点不同,它可识别并结合不同基因MAR区富含AT碱基序列从而调控基因复制、转录。本研究将检测SATB1在肝癌组织和细胞中的表达,研究其在肝癌侵袭转移中的作用机制。方法收集45例原发性肝癌及相应癌旁组织、选择8种不同转移潜能的肝癌细胞株,半定量PCR、western blot、免疫组化及免疫荧光检测SATB1 mRNA和蛋白水平表达,并用real-time PCR对SATB1 mRNA水平进行定量分析。采用shRNA干扰方法和真核表达SATB1全长基因下调或上调高、低转移潜能肝癌细胞株SATB1表达,而后进行以下实验：基因芯片技术筛选SATB1可调控的肝癌侵袭转移相关基因；real-time PCR、CHIP证实SATB1结合肝癌侵袭转移部分相关基因；倒置显微镜下观察细胞形态变化情况；鬼笔环肽标记细胞F-actin,采用激光共聚焦及免疫荧光技术检测细胞骨架重组情况；western blot检测细胞Vimentin、Tight junction protein 1、E-cadherin表达变化；transwell侵袭迁移实验检测细胞侵袭转移能力的改变；裸鼠皮下肿瘤生长实验及原位移植实验检测SATB1对肿瘤生长和转移的调控作用。结果45例原发性肝癌组织中,28(62%)例癌组织检测到SATB1蛋白水平表达,癌细胞核内可见棕黄色颗粒,癌组织中SATB1表达明显高于癌旁组织(18例,占40%,u=2.1, p<0.05), SATB1 mRNA水平定量分析结果显示,SATB1表达与肿瘤直径、分化程度、合并肝硬化以及癌组织转移有关(p<0.05),而与患者年龄、性别、肿瘤结节数无关。HBsAg阳性患者中肝癌组织SATB1 mRNA表达明显高于HBsAg阴性患者(p≤0.01)；8种不同转移潜能的肝癌细胞株中,高转移潜能的肝癌细胞SK-Hep-1、HCCLM8、MHCC-97H中SATB1 mRNA高表达(Ct值分别为2.83±0.6、2.72±0.54、2.09±0.28),中、低转移潜能肝癌细胞株MHCC-97L、SMMC-7721、HepG2、Huh7中SATB1mRNA低表达(Ct值分别为0.29±0.1、0.35±0.11、0.19±0.08、0.14±0.02,与高转移潜能的肝癌细胞相比,p<0.05),无转移能力Hep3B细胞中几乎无SATB1表达；高转移潜能SK-Hep-1细胞抑制SATB1后基因芯片技术检测结果表明,1450个基因表达上调2倍,1635个基因表达下调2倍；低转移潜能肝癌SMMC-7721细胞上调SATB1表达,1286个基因表达上调2倍,1463个基因表达下调2倍,两组结果对比,SATB1可调节肝癌侵袭转移基因有近200个,如粘附分子CDH3、FNl,信号转导基因TCF4,抑癌基因APC,蛋白酶MMP12、SLPI、CTSL1,转录因子Ets1,转移相关基因SERPINB5、VIL2等,对其中部分基因采用real-time PCR、CHIP实验方法研究,结果显示,SATB1可与这些基因MAR区相应位点结合并调控其表达；SK-Hep-1细胞抑制SATB1表达后,细胞形态明显改变,长纺锤体细胞收缩呈多角形,细胞规律方向性生长改为呈岛状增殖,鬼笔环肽标记细胞F-actin后,激光共聚焦显微镜下观察发现细胞骨架消失,横贯于细胞长轴的粗大应力纤维解聚成细小微丝,散布于细胞核周围,而SMMC-7721细胞上调SATB1表达后,椭圆形细胞拉长变为长纺锤体形,细胞间隙增宽,细胞长轴方向可见明显聚集的应力纤维,与抑制SATB1表达诱导细胞形态和骨架变化相反；抑制SATB1表达后,western blot检测细胞形态、骨架改变相关关键蛋白分子,发现Tight junction protein 1 (0.62±0.02 vs 0.06±0.01,p<0.01)、E-cadherin表达增强(0.82±0.04 vs 0.42±0.01, p<0.05), Vimentin表达下降(0.27±0.02vs 0.57±0.06,p<0.05),上调SATB1表达后,与对照相比,Tight junction protein 1 (0.35±0.01 vs 0.51±0.04, p<0.05)、E-cadherin表达下降(0.36±0.02 vs 0.64±0.06,p≤0.01), Vimentin表达增强(0.64±0.03 vs 0.34±0.04, p<0.05); transwell侵袭迁移实验结果表明,抑制SATB1表达,穿过基质胶的细胞较对照组明显减少(OD值0.16±0.02vs 0.33±0.05,p<0.05),迁移实验示穿过膜孔细胞较对照减少(OD值0.76±0.12 vs0.22±0.06,p<0.05)；上调SATB1表达,穿过基质胶的细胞明显增多,实验组与对照组相比p<0.05(0.67±0.08 vs 0.26±0.04);迁移实验示穿过膜孔细胞增多,实验组与对照组相比p<0.05(0.79±0.11 vs 0.26±0.04)；裸鼠皮下肿瘤生长实验表明,下调SATB1表达,实验组与对照组肿瘤平均重量分别为0.14±0.02g、0.43±0.06g(p<0.05),上调SATB1表达,实验组与对照组肿瘤平均质量分别为0.57±0.05g、0.2±0.07g(p<0.05)。肝癌组织原位移植(肿瘤组织接种到肝脏)实验检测SATB1上调或下调后对肿瘤侵袭转移能力的影响,因裸鼠在观察期内死亡,未观察到活体肿瘤侵袭转移方面的改变。结论SATB1在肝癌组织中高表达,其表达与肝癌组织远处转移有关,转移能力越强的肝癌细胞株,SATB1表达越高；下调或上调SATB1表达,可调控许多肝癌侵袭转移相关基因表达,SATB1可与这些基因MAR区相应位点结合并调控其表达,诱导细胞出现MET-EMT样的形态、骨架改变,并调控Vimentin、Tight junction protein 1、E-cadherin蛋白表达的增强或下降；下调或上调SATB1表达的综合效应为肝癌细胞株侵袭转移能力的降低或增强。体内实验表明,SATB1可调控肿瘤生长。更多还原

【Abstract】 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.更多还原