骨桥蛋白在肝癌侵袭转移中作用及其反义靶向治疗的实验研究
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摘要
肝细胞肝癌(hepatocellular carcinoma HCC，以下简称肝癌)在全球恶性肿瘤发病率中占第6位，恶性肿瘤死因中占第3位。在中国恶性肿瘤死因中肝癌占第2位。肝癌复发转移是目前肝癌治疗失败的主要原因。手术切除是肝癌最有效的治疗方法，但是肝癌根治性切除术后5年复发转移率为61.5％，即使是小肝癌也达43.5％。肝癌复发转移是多基因、多因素参与的复杂过程。探索肝癌复发转移的分子机制，寻找有效的靶向治疗是进一步提高肝癌治疗效果的关键。骨桥蛋白(osteopontin，OPN)是一种分泌型糖蛋白，能促进细胞趋化，粘附和迁移。以往的研究发现OPN与肝癌侵袭转移相关。但是，OPN促进肝癌侵袭转移的分子机制及其在肝癌侵袭转移中高表达机制尚未阐明。
第一部分骨桥蛋白在不同转移潜能肝癌细胞系及肝癌组织中表达情况
研究不同转移潜能人肝癌细胞系及其肝癌组织中OPN表达情况，分析OPN表达与肝癌细胞侵袭转移潜能、肝癌术后复发转移的关系。应用细胞免疫组化、半定量RT-PCR、Western Blot和ELISA检测不同转移潜能人肝癌细胞系SMMC-7721、MHCC97-L和HCCLM6 OPN表达情况。应用组织芯片、免疫组化检测200例肝癌组织中OPN表达。结果发现OPN表达水平随着肝癌细胞侵袭转移潜能增加逐渐升高。肝癌组织中OPN表达与患者性别、年龄、血清AFP水平、肿瘤大小、有无包膜、细胞分化以及肝门淋巴结转移无明显相关性，而与肝癌TNM分期、门静脉癌栓和术后复发转移发生有明显相关(P＜0.05)。术后有复发转移患者的肝癌组织中66％表达OPN，而术后无复发转移患者的肝癌组织中仅37％表达OPN，两者比较差异具有显著性(P＜0.05)。肝癌组织中OPN表达是预测肝癌术后是否发生复发转移的指标。
第二部分骨桥蛋白促进肝癌侵袭转移机制的初步研究
研究OPN对肝癌恶性表型影响及其可能机制。用pcDNA 3.1(-)／OPN质粒转染低侵袭性无转移能力肝癌细胞系SMMC-7721细胞，以空质粒转染为对照，用半定量RT-PCR、ELISA和Western-blot检测转染后细胞OPN表达水平，体外功能试验观察细胞转染前后恶性表型的变化。用G418筛选获得稳定高表达OPNSMMC-7721细胞克隆，以空质粒转染为对照。用肿瘤转移相关的基因芯片比较两者基因表达的差异。real-time PCR、ELISA验证有差异表达的MMP-2、uPA基因。结果发现SMMC-7721细胞转染OPN质粒后，OPN表达水平明显升高。体外功能试验显示SMMC-7721细胞转染OPN质粒后，细胞粘附、运动和侵袭能力明显升高(转染后细胞粘附率75.33％±10.59％vs 57.34％±2.52％，运动试验穿膜细胞数14.33±2.51 vs 6.34±1.53，侵袭试验穿膜细胞数8.23±1.53 vs 4.124±1.29，P均＜0.05)，而转染后，细胞增殖能力的改变不明显。基因芯片比较发现稳定高表达OPN SMMC-7721细胞有88个肿瘤转移相关基因表达上调，有6个肿瘤转移相关基因表达下调。real-time PCR、ELISA发现高表达OPN SMMC-7721细胞MMP-2、uPA表达水平明显高于对照组(P均＜0.05)，而MMP-9表达水平没有明显变化，与基因芯片结果符合。OPN可能是通过增加MMP-2、uPA等基因的表达促进肝癌侵袭转移。
第三部分高转移潜能肝癌细胞系HCCLM6细胞中骨桥蛋白高表达机制的初步研究
探讨促进高转移潜能肝癌细胞系HCCLM6细胞OPN表达的相关机制。(1)提取基因组DNA，PCR扩增OPN启动子(-1488～+185)，应用转录因子芯片比较不同转移潜能、不同OPN表达水平的肝癌细胞系SMMC-7721细胞CCLM6细胞中结合OPN启动子的转录因子活性差异，筛选出与OPN转录有关的、有活性差异的转录因子。用凝胶电泳迁移率变动分析(EMSA)验证有活性差异的转录因子c-myb。real-time PCR检测不同转移潜能肝癌细胞系转录因子c-myb mRNA表达水平。用小RNA干扰(siRNA)抑制HCCLM6细胞c-myb表达后，观察其对OPN表达、细胞体外侵袭能力的影响。结果发现在SMMC-7721细胞和HCCLM6细胞中，调控OPN的转录因子活性存在显著差异，筛选出23个转录因子活性与OPN转录有关。EMSA、real-time FCR验证SMMC-7721细胞和HCCLM6细胞中转录因子c-myb活性、mRNA表达水平存在显著差异(P＜0.05)。c-myb特异性siRNA可显著下调HCCLM6细胞c-myb、OPN表达，明显抑制其体外侵袭能力。(2)运用生物信息学方法预测可能调控OPN翻译过程microRNA(miRNA)，real-time PCR检测不同转移潜能肝癌细胞miRNA-96转录水平，用miRNA-96抑制剂阻断或模拟剂增加HCCLM6细胞miRNA-96水平，观察其对OPN表达、细胞体外侵袭能力的影响。miRNA-96转录水平在SMMC-7721细胞和HCCLM6细胞之间存在显著差异(P＜0.05)。HCCLM6细胞miRNA-96转录水平升高与OPN表达增加有关。抑制HCCLM6细胞中miRNA-96水平可显著下调其OPN表达，抑制其体外侵袭能力。
第四部分骨桥蛋白反义寡核苷酸靶向抑制肝癌侵袭转移的实验研究
观察2’-O-(2-甲氧乙烷基)(2’-MOE)修饰的OPN反义寡核苷酸(ASOs)对高转移潜能肝癌细胞系HCCLM6侵袭转移的靶向干预作用，并探讨其可能分子机制。用脂质体转染ASOs干预HCCLM6细胞后，细胞免疫组化、半定量RT-PCR、Western Blot和ELISA方法检测细胞OPN表达的改变，以随机序列寡核苷酸(ASOs-C)和单用脂质体为对照组。MTT、流式细胞仪、Trans-well细胞运动、侵袭试验观察对细胞增殖、细胞凋亡、运动、侵袭能力的影响。ELISA方法测定ASOs处理后，HCCLM6细胞分泌MMP-2、uPA的变化。36只HCCLM6细胞肝脏原位接种裸鼠，随机、平均分成OPN ASOs治疗组、ASOs-C和生理盐水对照组。在原位接种肿瘤后第2天开始给药，腹腔注射(ip．)ASOs 50mg／kg，隔两天注射一次，连续给药12次。至第6周末，每组随机选6只裸鼠解剖观察肝癌生长、转移情况，免疫组化检测肝癌组织中OPN蛋白表达，肺组织切片H．E．染色观察肺转移情况。全血行血常规检查，血清行肝功能指标检查。每组剩余6只裸鼠观察生存时间。结果发现OPN ASOs能明显抑制HCCLM6细胞OPN表达，而ASOs-C和单用脂质体对OPN表达无抑制作用(P＜0.05)。400nM OPN ASOs处理HCCLM6细胞24h后，能明显抑制其体外运动、侵袭能力，而且MMP-2、uPA分泌水平明显降低，与ASOs-C和单用脂质体处理组相比差异有显著性(P＜0.05)。400nM OPN ASOs处理HCCLM6细胞24h后，对细胞增殖、凋亡无明显作用。裸鼠体内试验发现OPN ASOs治疗组肝癌肺转移发生率(2／6)明显低于ASOs-C和生理盐水对照组的肺转移发生率(均为6／6)，但是各组荷瘤裸鼠原位肿瘤的大小、生存时间没有明显差别(P均＞0.05)。ASOs对荷瘤裸鼠血细胞和肝功能没有明显影响。OPN ASOs能抑制高转移潜能肝癌细胞系HCCLM6细胞体内、外侵袭转移能力，是潜在的抗肝癌复发转移的靶向药物。
结论
1)OPN表达和肝癌细胞侵袭转移能力相关；肝癌组织中OPN表达与肝癌TNM分期、门静脉癌栓、术后复发转移发生有关。
2)OPN促进低侵袭性肝癌细胞系SMMC-7721细胞的恶性表型，能引起多个肿瘤转移相关基因表达的改变。OPN通过增加MMP-2、uPA等基因的表达，促进肝癌恶性表型。
3)高转移潜能肝癌细胞系HCCLM6细胞中，转录因子c-myb与OPN表达相关。miRNA-96转录水平与HCCLM6细胞OPN表达有关。c-myb表达水平、miRNA-96转录水平的增加，促进HCCLM6细胞OPN表达，两者可以作为调控OPN表达潜在的靶点。
4)靶向OPN反义寡核苷酸能抑制高转移潜能肝癌细胞系HCCLM6细胞体内、外侵袭转移能力，是潜在抗肝癌复发转移的靶向药物。
潜在临床应用价值
1)OPN是抑制肝癌复发转移的潜在治疗靶点。
2)c-myb、miRNA-96可以作为调控肝癌OPN表达的靶点。
创新点
1)发现OPN促进肝癌恶性表型中，MMP-2、uPA表达增加起重要作用。
2)发现c-myb、miRNA-96水平增加可以促进肝癌细胞OPN表达。
3)发现靶向OPN反义寡核苷酸可以抑制肝癌侵袭转移。
Hepatocellular carcinoma (HCC) is the sixth prevalent malignant tumor, which is ranked the third of tumor-related death in the world and is the second in China. HCC recurrence and metastasis lead to the failure of treatment. Surgery remains the best treatment for HCC. However, the rate of HCC recurrence and metastasis is 61．5% after radical resection and is 43．5% even for small HCC. Multi-genes and multi-factors are involved in the processes of HCC recurrence and metastasis. Elucidating molecular mechanisms of HCC recurrence/metastasis and searching for effective target therapies are the promising pathway to improve the survival. OPN is the secreted glycoprotein promoting cell chemotaxis, adhesion and migration. Previous studies indicate OPN expression is associated with HCC progression. However, the mechanisms of OPN promoting HCC recurrence/metastasis and which factors increase HCC OPN expression are poorly understood.Part one: Osteopontin expression in hepatocellular carcinoma (HCC)cell lines with different metastatic potential and HCC samples
To study the relationships between OPN expression and HCC cell lines with different metastatic potential and HCC recurrence/metastasis after surgery. Immunocytochemistry, RT-PCR, Western Blot and ELISA were applied to detect OPN expression of HCC cell lines SMMC-7721, MHCC97-L and HCCLM6 with different metastastic potential. Immunohistochemistry of OPN was performed in the tissue arrays containing 200 HCC samples. We found that OPN expression was leveled with increasing metastatic potential of cell lines. OPN expression of samples was not associated with age, sex, serum AFP level, tumor size, cell differentiation, tumor capsule, lymphatic node metastasis while it was significantly associated with HCC TNM classification, portal vein tumor thrombi and HCC recurrence/metastasis after surgery. OPN expression detected in 66% HCC samples of patients with recurrence/metastasis after surgery was significantly higher than 37% samples of patients without recurrence /metastasis after surgery(P<0．05) . OPN expression is the indicator of HCC recurrence/metastasis after surgery.Part two: Mechanisms of osteopontin promoting hepatocellular carcinomainvasion and metastasis
To study the mechanisms of OPN promoting HCC malignant phenotypes. HCC cell line SMMC-7721 cells with low invasion and no metastatic potential were transfected by pcDNA 3．1(-)/OPN plasmid while cells transfected with mock plasmid were controlled group. OPN expression was testified by RT-PCR, Western Blot and ELISA. Functional assays in vitro were performed to observe changes of malignant phenotypes after transfection. SMMC-7721 cells stably expressing high level of OPN were established by plasmid transfection and G418 screening while cells transfected with mock plasmid were used as control. Differences of metastasis-related genes expression between two groups were compared by gene chips. Real-time PCR and ELISA testified the different expression of MMP-2 and uPA in the gene chips. OPN expression of SMMC-7721 cells was elevated after transient transfection. Functional assays in vitro indicated that SMMC-7721 cells after transfection showed higher adhesive, migrant and invasive capabilities(cell adhesion rate: 75．33%±10．59% vs 57．34%±2．52%; cells of outer surface in migrant assay: 14．33±2．51 vs 6．34±1．53; cells in invasive assay: 8．23±1．53 vs 4．12±1．29) while the ability of cell proliferation was similar. 88 metastasis-related genes were up-regulated in SMMC-7721 cells with high OPN expression while 6 genes were down-regulated. Real-time PCR and ELISA found the level of MMP-2 and uPA expression were higher than the controlled group while MMP-9 level did not increase. OPN might enhance the expression levels of MMP-2, uPA and other genes to promote malignant phenotypes of SMMC-7721 cells.Part three: Mechanisms of osteopontin high expression in hepatocellular carcinoma cell line HCCLM6 with high metastatic potential.
To study the relative mechanisms of OPN high expression in HCC cell line HCCLM6 with high metastatic potential. Firstly, Genomic DNA was extracted and OPN promoter amplified by PCR was applied as probe. Using Protein/DNA array, transcription factor activity profiles of binding with OPN promoter in cell lines SMMC-7721 and HCCLM6 of different OPN expression, different metastatic potential were examined. 23 different transcription factors activities were associated with cell line OPN expression. Transcription factor c-myb activity was confirmed by electrophoretic mobility shift assays (EMSA) in the two cell lines. Real-time PCR detected c-myb mRNA expression in cell lines with different OPN expression levels. Small interference RNA (siRNA) inhibited c-myb expression. We found that c-myb siRNA reduced expression level of c-myb and OPN in HCCLM6 cells and also inhibited HCCLM6 cells invasive ability in vitro. Secondly, using informatics, we found that microRNA(miRAN)-96 maybe participate regulating OPN translation processes. Real-time PCR examined the transcriptional level of miRNA-96 in cell lines. OPN expression was determined after increasing or decreasing miRNA-16 level by miRNA-96 mimics and miRNA-96 inhibitor in HCCLM6 cells. We found that miRNA-16 level was different in cell lines with different metastatic potential. OPN expression was associated with miRNA-96 level in HCCLM6 cells. Decreasing miRNA-96 level significantly suppressed OPN expression and inhibited the invasive ability of HCCLM6 cells in vitro.Part four: Antisene oligonucleotides targeting osteopontin suppress hepatocellular carcinoma invasion and metastasis
To evaluate the effects of 2'-O-(2-methoxyethyl)-modified antisense oligonucleotides (ASOs) targeting OPN on HCC invasion and metastasis and study the underlying mechanisms. ASOs were delivered to HCC cell line HCCLM6 with high metastatic potential in the form of complexes with Lipofectamine. HCCLM6 cells were treated with OPN ASOs while base-randomized oligonucleotides (ASOs-C) and Lipofectamine alone were applied as controls. OPN expression was detected by RT-PCR, Western blot and ELISA. MTT, Flow cytometer, trans-well migrant/invasive assays were performed after HCCLM6 cells were treated with ASOs. MMP-2 and uPA level were measured by ELISA. 36 nude mice with orthotropic implantation of HCCLM6 cells were randomly equally assigned to three groups as the following: OPN ASOs treatment group, ASOs-C controlled group and injection of physiological saline(NS) group. Drug administration was started at the second day after tumor implantation. 50mg/kg OPN ASOs, ASOs-C or 0．2ml NS was injected through intraperitoneum(i.p.) once every three days for 12 times. After six weeks, 6 mice randomly selected from each experimental group were sacrificed to observe the growth and metastasis of HCCLM6．OPN expression was assessed by immunohistochemistry. Pulmonary metastases were detected by H.E. stains. Blood samples were collected for routine blood analysis and detecting AST, ALT level in serum. Another 6 mice in each group were maintained to evaluate life span. We found that in vitro, expression of OPN in HCCLM6 cells was significantly suppressed by OPN ASOs while ASOs-C and Lipofectamine did not affect(P<0．05) . HCCLM6 cells treated by 400 nM OPN ASO for 24h showed in significantly decreased migrant and invasive abilities. The same concentration of OPN ASOs also significantly reduced the levels of MMP2 and uPA expression. OPN ASOs did not affect HCCLM6 cells growth or promote cell apoptosis. The incidence of lung metastasis of HCCLM6 treated by OPN ASO in nude mice was significantly inhibited (2/6) compared with the groups treated by ASOs-C(6/6) and NS(6/6) . The tumor weight and the survival of nude mice with HCCLM6 were similar among three groups. ASOs had no apparent hepatic and blood toxicities. OPN ASOs may serve as a novel therapeutic strategy for inhibiting HCC invasion and metastasis.Conclusions
1．OPN expression is associated with the invasive and metastatic abilities of HCC cell lines. OPN expression is associated with HCC TNM classification, portal vein tumor thrombi, HCC recurrence/metastasis after surgery.
2．OPN promotes malignant phenotypes of SMMC-7721 cells with low invasive capability. Increasing OPN expression results in changes of many tumor metastasis-related genes. OPN may increase the expression level of MMP-2, uPA and other genes to enhance the malignant phenotypes of HCC.
3．Transcription factor c-myb is correlated with OPN expression in HCCLM6 cells with high metastatic potential. MiRNA-96 level is also associated with OPN expression. Increasing c-myb or miRNA-96 level promote the expression of OPN, underlying new targets of regulating OPN expression.
4．Targeting OPN antisense oligonucleotides significantly inhibit the invasion and metastasis of HCCLM6 with high metastatic potential, implicating a novel drug for anti-HCC recurrence/metastasis. The potential application of this work
1．OPN is the new therapeutic target of anti-recurrence/metastasis for HCC.
2．c-myb or miRNA-96 maybe the new targets of regulating OPN expression.The novelty of this work
1．Demonstrate that OPN increases MMP-2, uPA and other genes expression to promote HCC malignant phenotypes.
2．Explore the mechanisms of OPN high expression and find that c-myb and miRNA-96 promote OPN expression in HCC.
3．Find that targeting OPN antisense oligonucleotides inhibit HCC invasion and metastasis.
第一部分骨桥蛋白在不同转移潜能肝癌细胞系及肝癌组织中表达情况
研究不同转移潜能人肝癌细胞系及其肝癌组织中OPN表达情况，分析OPN表达与肝癌细胞侵袭转移潜能、肝癌术后复发转移的关系。应用细胞免疫组化、半定量RT-PCR、Western Blot和ELISA检测不同转移潜能人肝癌细胞系SMMC-7721、MHCC97-L和HCCLM6 OPN表达情况。应用组织芯片、免疫组化检测200例肝癌组织中OPN表达。结果发现OPN表达水平随着肝癌细胞侵袭转移潜能增加逐渐升高。肝癌组织中OPN表达与患者性别、年龄、血清AFP水平、肿瘤大小、有无包膜、细胞分化以及肝门淋巴结转移无明显相关性，而与肝癌TNM分期、门静脉癌栓和术后复发转移发生有明显相关(P＜0.05)。术后有复发转移患者的肝癌组织中66％表达OPN，而术后无复发转移患者的肝癌组织中仅37％表达OPN，两者比较差异具有显著性(P＜0.05)。肝癌组织中OPN表达是预测肝癌术后是否发生复发转移的指标。
第二部分骨桥蛋白促进肝癌侵袭转移机制的初步研究
研究OPN对肝癌恶性表型影响及其可能机制。用pcDNA 3.1(-)／OPN质粒转染低侵袭性无转移能力肝癌细胞系SMMC-7721细胞，以空质粒转染为对照，用半定量RT-PCR、ELISA和Western-blot检测转染后细胞OPN表达水平，体外功能试验观察细胞转染前后恶性表型的变化。用G418筛选获得稳定高表达OPNSMMC-7721细胞克隆，以空质粒转染为对照。用肿瘤转移相关的基因芯片比较两者基因表达的差异。real-time PCR、ELISA验证有差异表达的MMP-2、uPA基因。结果发现SMMC-7721细胞转染OPN质粒后，OPN表达水平明显升高。体外功能试验显示SMMC-7721细胞转染OPN质粒后，细胞粘附、运动和侵袭能力明显升高(转染后细胞粘附率75.33％±10.59％vs 57.34％±2.52％，运动试验穿膜细胞数14.33±2.51 vs 6.34±1.53，侵袭试验穿膜细胞数8.23±1.53 vs 4.124±1.29，P均＜0.05)，而转染后，细胞增殖能力的改变不明显。基因芯片比较发现稳定高表达OPN SMMC-7721细胞有88个肿瘤转移相关基因表达上调，有6个肿瘤转移相关基因表达下调。real-time PCR、ELISA发现高表达OPN SMMC-7721细胞MMP-2、uPA表达水平明显高于对照组(P均＜0.05)，而MMP-9表达水平没有明显变化，与基因芯片结果符合。OPN可能是通过增加MMP-2、uPA等基因的表达促进肝癌侵袭转移。
第三部分高转移潜能肝癌细胞系HCCLM6细胞中骨桥蛋白高表达机制的初步研究
探讨促进高转移潜能肝癌细胞系HCCLM6细胞OPN表达的相关机制。(1)提取基因组DNA，PCR扩增OPN启动子(-1488～+185)，应用转录因子芯片比较不同转移潜能、不同OPN表达水平的肝癌细胞系SMMC-7721细胞CCLM6细胞中结合OPN启动子的转录因子活性差异，筛选出与OPN转录有关的、有活性差异的转录因子。用凝胶电泳迁移率变动分析(EMSA)验证有活性差异的转录因子c-myb。real-time PCR检测不同转移潜能肝癌细胞系转录因子c-myb mRNA表达水平。用小RNA干扰(siRNA)抑制HCCLM6细胞c-myb表达后，观察其对OPN表达、细胞体外侵袭能力的影响。结果发现在SMMC-7721细胞和HCCLM6细胞中，调控OPN的转录因子活性存在显著差异，筛选出23个转录因子活性与OPN转录有关。EMSA、real-time FCR验证SMMC-7721细胞和HCCLM6细胞中转录因子c-myb活性、mRNA表达水平存在显著差异(P＜0.05)。c-myb特异性siRNA可显著下调HCCLM6细胞c-myb、OPN表达，明显抑制其体外侵袭能力。(2)运用生物信息学方法预测可能调控OPN翻译过程microRNA(miRNA)，real-time PCR检测不同转移潜能肝癌细胞miRNA-96转录水平，用miRNA-96抑制剂阻断或模拟剂增加HCCLM6细胞miRNA-96水平，观察其对OPN表达、细胞体外侵袭能力的影响。miRNA-96转录水平在SMMC-7721细胞和HCCLM6细胞之间存在显著差异(P＜0.05)。HCCLM6细胞miRNA-96转录水平升高与OPN表达增加有关。抑制HCCLM6细胞中miRNA-96水平可显著下调其OPN表达，抑制其体外侵袭能力。
第四部分骨桥蛋白反义寡核苷酸靶向抑制肝癌侵袭转移的实验研究
观察2’-O-(2-甲氧乙烷基)(2’-MOE)修饰的OPN反义寡核苷酸(ASOs)对高转移潜能肝癌细胞系HCCLM6侵袭转移的靶向干预作用，并探讨其可能分子机制。用脂质体转染ASOs干预HCCLM6细胞后，细胞免疫组化、半定量RT-PCR、Western Blot和ELISA方法检测细胞OPN表达的改变，以随机序列寡核苷酸(ASOs-C)和单用脂质体为对照组。MTT、流式细胞仪、Trans-well细胞运动、侵袭试验观察对细胞增殖、细胞凋亡、运动、侵袭能力的影响。ELISA方法测定ASOs处理后，HCCLM6细胞分泌MMP-2、uPA的变化。36只HCCLM6细胞肝脏原位接种裸鼠，随机、平均分成OPN ASOs治疗组、ASOs-C和生理盐水对照组。在原位接种肿瘤后第2天开始给药，腹腔注射(ip．)ASOs 50mg／kg，隔两天注射一次，连续给药12次。至第6周末，每组随机选6只裸鼠解剖观察肝癌生长、转移情况，免疫组化检测肝癌组织中OPN蛋白表达，肺组织切片H．E．染色观察肺转移情况。全血行血常规检查，血清行肝功能指标检查。每组剩余6只裸鼠观察生存时间。结果发现OPN ASOs能明显抑制HCCLM6细胞OPN表达，而ASOs-C和单用脂质体对OPN表达无抑制作用(P＜0.05)。400nM OPN ASOs处理HCCLM6细胞24h后，能明显抑制其体外运动、侵袭能力，而且MMP-2、uPA分泌水平明显降低，与ASOs-C和单用脂质体处理组相比差异有显著性(P＜0.05)。400nM OPN ASOs处理HCCLM6细胞24h后，对细胞增殖、凋亡无明显作用。裸鼠体内试验发现OPN ASOs治疗组肝癌肺转移发生率(2／6)明显低于ASOs-C和生理盐水对照组的肺转移发生率(均为6／6)，但是各组荷瘤裸鼠原位肿瘤的大小、生存时间没有明显差别(P均＞0.05)。ASOs对荷瘤裸鼠血细胞和肝功能没有明显影响。OPN ASOs能抑制高转移潜能肝癌细胞系HCCLM6细胞体内、外侵袭转移能力，是潜在的抗肝癌复发转移的靶向药物。
结论
1)OPN表达和肝癌细胞侵袭转移能力相关；肝癌组织中OPN表达与肝癌TNM分期、门静脉癌栓、术后复发转移发生有关。
2)OPN促进低侵袭性肝癌细胞系SMMC-7721细胞的恶性表型，能引起多个肿瘤转移相关基因表达的改变。OPN通过增加MMP-2、uPA等基因的表达，促进肝癌恶性表型。
3)高转移潜能肝癌细胞系HCCLM6细胞中，转录因子c-myb与OPN表达相关。miRNA-96转录水平与HCCLM6细胞OPN表达有关。c-myb表达水平、miRNA-96转录水平的增加，促进HCCLM6细胞OPN表达，两者可以作为调控OPN表达潜在的靶点。
4)靶向OPN反义寡核苷酸能抑制高转移潜能肝癌细胞系HCCLM6细胞体内、外侵袭转移能力，是潜在抗肝癌复发转移的靶向药物。
潜在临床应用价值
1)OPN是抑制肝癌复发转移的潜在治疗靶点。
2)c-myb、miRNA-96可以作为调控肝癌OPN表达的靶点。
创新点
1)发现OPN促进肝癌恶性表型中，MMP-2、uPA表达增加起重要作用。
2)发现c-myb、miRNA-96水平增加可以促进肝癌细胞OPN表达。
3)发现靶向OPN反义寡核苷酸可以抑制肝癌侵袭转移。
Hepatocellular carcinoma (HCC) is the sixth prevalent malignant tumor, which is ranked the third of tumor-related death in the world and is the second in China. HCC recurrence and metastasis lead to the failure of treatment. Surgery remains the best treatment for HCC. However, the rate of HCC recurrence and metastasis is 61．5% after radical resection and is 43．5% even for small HCC. Multi-genes and multi-factors are involved in the processes of HCC recurrence and metastasis. Elucidating molecular mechanisms of HCC recurrence/metastasis and searching for effective target therapies are the promising pathway to improve the survival. OPN is the secreted glycoprotein promoting cell chemotaxis, adhesion and migration. Previous studies indicate OPN expression is associated with HCC progression. However, the mechanisms of OPN promoting HCC recurrence/metastasis and which factors increase HCC OPN expression are poorly understood.Part one: Osteopontin expression in hepatocellular carcinoma (HCC)cell lines with different metastatic potential and HCC samples
To study the relationships between OPN expression and HCC cell lines with different metastatic potential and HCC recurrence/metastasis after surgery. Immunocytochemistry, RT-PCR, Western Blot and ELISA were applied to detect OPN expression of HCC cell lines SMMC-7721, MHCC97-L and HCCLM6 with different metastastic potential. Immunohistochemistry of OPN was performed in the tissue arrays containing 200 HCC samples. We found that OPN expression was leveled with increasing metastatic potential of cell lines. OPN expression of samples was not associated with age, sex, serum AFP level, tumor size, cell differentiation, tumor capsule, lymphatic node metastasis while it was significantly associated with HCC TNM classification, portal vein tumor thrombi and HCC recurrence/metastasis after surgery. OPN expression detected in 66% HCC samples of patients with recurrence/metastasis after surgery was significantly higher than 37% samples of patients without recurrence /metastasis after surgery(P<0．05) . OPN expression is the indicator of HCC recurrence/metastasis after surgery.Part two: Mechanisms of osteopontin promoting hepatocellular carcinomainvasion and metastasis
To study the mechanisms of OPN promoting HCC malignant phenotypes. HCC cell line SMMC-7721 cells with low invasion and no metastatic potential were transfected by pcDNA 3．1(-)/OPN plasmid while cells transfected with mock plasmid were controlled group. OPN expression was testified by RT-PCR, Western Blot and ELISA. Functional assays in vitro were performed to observe changes of malignant phenotypes after transfection. SMMC-7721 cells stably expressing high level of OPN were established by plasmid transfection and G418 screening while cells transfected with mock plasmid were used as control. Differences of metastasis-related genes expression between two groups were compared by gene chips. Real-time PCR and ELISA testified the different expression of MMP-2 and uPA in the gene chips. OPN expression of SMMC-7721 cells was elevated after transient transfection. Functional assays in vitro indicated that SMMC-7721 cells after transfection showed higher adhesive, migrant and invasive capabilities(cell adhesion rate: 75．33%±10．59% vs 57．34%±2．52%; cells of outer surface in migrant assay: 14．33±2．51 vs 6．34±1．53; cells in invasive assay: 8．23±1．53 vs 4．12±1．29) while the ability of cell proliferation was similar. 88 metastasis-related genes were up-regulated in SMMC-7721 cells with high OPN expression while 6 genes were down-regulated. Real-time PCR and ELISA found the level of MMP-2 and uPA expression were higher than the controlled group while MMP-9 level did not increase. OPN might enhance the expression levels of MMP-2, uPA and other genes to promote malignant phenotypes of SMMC-7721 cells.Part three: Mechanisms of osteopontin high expression in hepatocellular carcinoma cell line HCCLM6 with high metastatic potential.
To study the relative mechanisms of OPN high expression in HCC cell line HCCLM6 with high metastatic potential. Firstly, Genomic DNA was extracted and OPN promoter amplified by PCR was applied as probe. Using Protein/DNA array, transcription factor activity profiles of binding with OPN promoter in cell lines SMMC-7721 and HCCLM6 of different OPN expression, different metastatic potential were examined. 23 different transcription factors activities were associated with cell line OPN expression. Transcription factor c-myb activity was confirmed by electrophoretic mobility shift assays (EMSA) in the two cell lines. Real-time PCR detected c-myb mRNA expression in cell lines with different OPN expression levels. Small interference RNA (siRNA) inhibited c-myb expression. We found that c-myb siRNA reduced expression level of c-myb and OPN in HCCLM6 cells and also inhibited HCCLM6 cells invasive ability in vitro. Secondly, using informatics, we found that microRNA(miRAN)-96 maybe participate regulating OPN translation processes. Real-time PCR examined the transcriptional level of miRNA-96 in cell lines. OPN expression was determined after increasing or decreasing miRNA-16 level by miRNA-96 mimics and miRNA-96 inhibitor in HCCLM6 cells. We found that miRNA-16 level was different in cell lines with different metastatic potential. OPN expression was associated with miRNA-96 level in HCCLM6 cells. Decreasing miRNA-96 level significantly suppressed OPN expression and inhibited the invasive ability of HCCLM6 cells in vitro.Part four: Antisene oligonucleotides targeting osteopontin suppress hepatocellular carcinoma invasion and metastasis
To evaluate the effects of 2'-O-(2-methoxyethyl)-modified antisense oligonucleotides (ASOs) targeting OPN on HCC invasion and metastasis and study the underlying mechanisms. ASOs were delivered to HCC cell line HCCLM6 with high metastatic potential in the form of complexes with Lipofectamine. HCCLM6 cells were treated with OPN ASOs while base-randomized oligonucleotides (ASOs-C) and Lipofectamine alone were applied as controls. OPN expression was detected by RT-PCR, Western blot and ELISA. MTT, Flow cytometer, trans-well migrant/invasive assays were performed after HCCLM6 cells were treated with ASOs. MMP-2 and uPA level were measured by ELISA. 36 nude mice with orthotropic implantation of HCCLM6 cells were randomly equally assigned to three groups as the following: OPN ASOs treatment group, ASOs-C controlled group and injection of physiological saline(NS) group. Drug administration was started at the second day after tumor implantation. 50mg/kg OPN ASOs, ASOs-C or 0．2ml NS was injected through intraperitoneum(i.p.) once every three days for 12 times. After six weeks, 6 mice randomly selected from each experimental group were sacrificed to observe the growth and metastasis of HCCLM6．OPN expression was assessed by immunohistochemistry. Pulmonary metastases were detected by H.E. stains. Blood samples were collected for routine blood analysis and detecting AST, ALT level in serum. Another 6 mice in each group were maintained to evaluate life span. We found that in vitro, expression of OPN in HCCLM6 cells was significantly suppressed by OPN ASOs while ASOs-C and Lipofectamine did not affect(P<0．05) . HCCLM6 cells treated by 400 nM OPN ASO for 24h showed in significantly decreased migrant and invasive abilities. The same concentration of OPN ASOs also significantly reduced the levels of MMP2 and uPA expression. OPN ASOs did not affect HCCLM6 cells growth or promote cell apoptosis. The incidence of lung metastasis of HCCLM6 treated by OPN ASO in nude mice was significantly inhibited (2/6) compared with the groups treated by ASOs-C(6/6) and NS(6/6) . The tumor weight and the survival of nude mice with HCCLM6 were similar among three groups. ASOs had no apparent hepatic and blood toxicities. OPN ASOs may serve as a novel therapeutic strategy for inhibiting HCC invasion and metastasis.Conclusions
1．OPN expression is associated with the invasive and metastatic abilities of HCC cell lines. OPN expression is associated with HCC TNM classification, portal vein tumor thrombi, HCC recurrence/metastasis after surgery.
2．OPN promotes malignant phenotypes of SMMC-7721 cells with low invasive capability. Increasing OPN expression results in changes of many tumor metastasis-related genes. OPN may increase the expression level of MMP-2, uPA and other genes to enhance the malignant phenotypes of HCC.
3．Transcription factor c-myb is correlated with OPN expression in HCCLM6 cells with high metastatic potential. MiRNA-96 level is also associated with OPN expression. Increasing c-myb or miRNA-96 level promote the expression of OPN, underlying new targets of regulating OPN expression.
4．Targeting OPN antisense oligonucleotides significantly inhibit the invasion and metastasis of HCCLM6 with high metastatic potential, implicating a novel drug for anti-HCC recurrence/metastasis. The potential application of this work
1．OPN is the new therapeutic target of anti-recurrence/metastasis for HCC.
2．c-myb or miRNA-96 maybe the new targets of regulating OPN expression.The novelty of this work
1．Demonstrate that OPN increases MMP-2, uPA and other genes expression to promote HCC malignant phenotypes.
2．Explore the mechanisms of OPN high expression and find that c-myb and miRNA-96 promote OPN expression in HCC.
3．Find that targeting OPN antisense oligonucleotides inhibit HCC invasion and metastasis.
引文
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1. Denhardt DT, Guo X. Osteopontin:a protein with diverse functions. FASEB J, 1993, 7: 1574-1482.
2. Rittling SR, Chambers AF. Role of osteopontin in tumour progression. Br J Cancer, 2004, 90: 1877-1881.
3. Wai PY, Kuo PC. The role of Osteopontin in tumor metastasis. J Surg Res, 2004, 121: 228-241.
4. Chakraborty G, Jain S, Behera R, et al. The multifaceted role of osteopontin in cell signaling, tumor progression and angiogenesis. Curr Mol Med, 2006, 6:819-830.
5. Rangaswami H, Bulbule A, Kundu GC. Osteopontin: role in cell signaling and cancer progression. Trends Cell Biol. 2006, 16:79-87
6. Coppola D, Szabo M, Boulware D, et al. Correlation of osteopontin protein expression and pathological stage across a wide variety of tumor histologies. Clin Cancer Res, 2004,10:184-190.
7. Yeatman TJ, Chamers AF. Osteopontin and colon cancer progression. Clin Cancer Res, 2003, 20, 85-90.
8. Das R, Mahabeleshwar GH, Kundu GC. Osteopontin induces AP-1-mediated secretion of urokinase-type plasminogen activator through c-Src-dependent epidermal growth factor receptor transactivation in breast cancer cells. J Biol Chem, 2004, 279:1051-11064.
9. Philip S, Bulbule A, Kundu GC. Osteopontin stimulates tumor growth and activation ofpromatrix metalloproteinase-2 through nuclear factor-kappa B-mediated induction of membrane type 1 matrix metalloproteinase in murine melanoma cells. J Biol Chem, 2001, 276: 44926-44935.
10. Jain S, Chakraborty G, Kundu GC. The crucial role of cyclooxygenase-2 in osteopontin-induced protein kinase C alpha/c-Src/IkappaB kinase alpha/beta-dependent prostate tumor progression and angiogenesis. Cancer Res, 2006,66: 6638-6648.
11. Cook AC, Chambers AF, Turley EA, et al. Osteopontin induction of hyaluronan synthase 2 expression promotes breast cancer malignancy. J Biol Chem, 2006, 281:24381-24389.
12. Gotoh M, Sakamoto M, Kanetaka K, et al. Overexpression of osteopontin in hepatocellular carcinoma. Pathol Int, 2002,52:19-24.
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14. Ye QH, Qin LX, Forgues M, et al. Predicting hepatitis B virus-positive metastatic hepatocellular carcinomas using gene expression profiling and supervised machine learning. Nat Med, 2003, 9: 416-423.
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14. Jain S, Chakraborty G, Kundu GC. The crucial role of cyclooxygenase-2 in osteopontin-induced protein kinase C alpha/c-Src/IkappaB kinase alpha/ beta-dependent prostate tumor progression and angiogenesis. Cancer Res, 2006,66:6638-6648.
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1. Denhardt DT, Guo X. Osteopontin: a protein with diverse functions. FASEB J, 1993, 7: 1574-1482.
2. Rittling SR, Chambers AF. Role of osteopontin in tumour progression. Br J Cancer, 2004, 90: 1877-1881.
3. Wai PY, Kuo PC. The role of Osteopontin in tumor metastasis. J Surg Res, 2004, 121: 228-241.
4. Chakraborty G, Jain S, Behera R, et al. The multifaceted role of osteopontin in cell signaling, tumor progression and angiogenesis. Curr Mol Med, 2006, 6: 819-830.
5. Rangaswami H, Bulbule A, Kundu GC. Osteopontin: role in cell signaling and cancer progression. Trends Cell Biol, 2006, 16: 79-87.
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12. Liu YN, Kang BB, Chen JH. Transcriptional regulation of human osteopontin promoter by C/EBPalpha and AML-1 in metastatic cancer cells. Oncogene. 2004, 23:278-88.
13. El-Tanani M, Barraclough R, Wilkinson MC, et al. Metastasis-inducing DNA regulates the expression of the osteopontin gene by binding the transcription factor Tcf-4. Cancer Res, 2001,61:5619-5629.
14. El-Tanani M, Platt-Higgins A, Rudland PS, et al. Ets gene PEA3 cooperates with beta-catenin-Lef-1 and c-Jun in regulation of osteopontin transcriptiort J Biol Chem, 2004, 279: 20794-20806.
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18. Gao C, Mi Z, Guo H, et al. A transcriptional repressor of osteopontin expression in the 4T1 murine breast cancer cell line. Biochem Biophy Res Commun, 2004, 321: 1010-1016.
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25. Tuck AB, Hota C, Wilson SM, et al. Osteopontin-induced migration of human mammary epithelial cells involves activation of EGF receptor and multiple signal transduction pathways. Oncogene, 2003, 22:1198-1205.
26. Das R, Mahabeleshwar GH, Kundu GC. Osteopontin stimulates cell motility and nuclear factor kappaB-mediated secretion of urokinase type plasminogen activator through phosphatidylinositol 3-kinase/Akt signaling pathways in breast cancer cells. J Biol Chem, 2003, 278: 28593-28606.
27. Das R, Mahabeleshwar GH, Kundu GC. Osteopontin induces A.P-1-mediated secretion of urokinase-type plasminogen activator through c-Src-dependent epidermal growth factor receptor transactivation in breast cancer cells. J Biol Chem, 2004,279:11051-11064.
28. Philip S, Kundu GC. Osteopontin induces nuclear factor kappa B-mediated promatrix metalloproteinase-2 activation through I kappa B alpha/IKK signaling pathways, and curcumin (diferulolylmethane) down-regulates these pathways. J Biol Chem, 2003, 278:14487-14497.
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46. Tang ZY, Ye SL, Liu YK, et al. A decade's studies on metastasis of hepatocellular carcinoma. J Cancer Res Clin Oncol. 2004, 130:187-196.
47. Zhang GX, Zhao ZQ, Wang HD, et al. Enhancement of osteopontin expression in HepG2 cells by epidermal growth factor via phosphatidylinositol 3-kinase signaling pathway. World J Gastroenterol. 2004; 10: 205-208.
48. Ye QH, Qin LX, Forgues M, et al. Predicting hepatitis B virus-positive metastatic hepatocellular carcinomas using gene expression profiling and supervised machine learning. Nat Med, 2003, 9: 416-423.
49. Gotoh M, Sakamoto M, Kanetaka K, et al. Overexpression of osteopontin in hepatocellular carcinoma. Pathol Int, 2002,52:19-24.
50. Pan HW, Ou YH, Peng SY, et al. Overexpression of osteopontin is associated with intrahepatic metastasis, early recurrence, and poorer prognosis of surgically resected hepatocellular carcinoma. Cancer, 2003, 98: 19-127.
51. Peng SY, Ou YH, Chen WJ, et al. Aberrant expressions of annexin A10 short isoform, osteopontin and alpha-fetoprotein at chromosome 4q cooperatively contribute to progression and poor prognosis of hepatocellular carcinoma. Int J Oncol, 2005, 26: 1053-1061.
52. Iso Y, Sawada T, Okada T, et al. Loss of E-cadherin mRNA and gain of osteopontin mRNA are useful markers for detecting early recurrence of HCV-related hepatocellular carcinoma. J Surg Oncol, 2005, 92: 304-311.
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2．张思维，李连弟，鲁风珠，等，中国1990-1992年原发性肝癌死亡调查分析．中华肿瘤杂志，1999，21：245-249．
3．汤钊猷．概述．见：汤钊猷主编．肿瘤转移复发的基础与临床．上海科技教育出版社，2003，1-24．
4. Tang ZY, Ye SL, Liu YK, et al. A decade's studies on metastasis of hepatocellular carcinoma. J Cancer Res Clin Oncol, 2004, 130: 187-196.
5. Tang ZY. Hepatocellular carcinoma surgery—review of the past and prospects for the 21st century. J Surg Oncol, 2005, 91: 95-96.
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9. Denhardt DT, Guo X. Osteopontin: a protein with diverse functions. FASEB J, 1993, 7:1574-1482.
10. Rittling SR, Chambers AF. Role ofosteopontin in tumor progression. Br J Cancer, 2004, 90: 1877-1881.
11. Wai PY, Kuo PC. The role of Osteopontin in tumor metastasis. J Surg Res, 2004, 121(2): 228-241
12. Chakraborty G, Jain S, Behera R, et al. The multifaceted role of osteopontin in cell signaling, tumor progression and angiogenesis. Curr Mol Med, 2006, 6:819-830.
13. Rangaswami H, Bulbule A, Kundu GC. Osteopontin: role in cell signaling and cancer progression. Trends Cell Biol, 2006, 16: 79-87.
14. Wu Y, Denhardt DT, and Rittling SR. Osteopontin is required for full expression of the transformed phenotype by the ras oncogene. Br J Cancer, 2000, 83, 156-163.
15. Behrend El, Craig AM, Wilson SM, et al. Reduced malignancy of rastransformed NIH 3T3 cells expressing antisense osteopontin RNA. Cancer Res, 1994, 54, 832-837
16. Coppola D, Szabo M, Boulware D et al. Correlation of osteopontin protein expression and pathological stage across a wide variety of tumor histologies. Clin Cancer Res, 2004,10:184-190.
17. Yeatman TJ, Charners AF. Osteopontin and colon cancer progression. Clin Cancer Res, 2003, 20, 85-90.
18. Koopmann J, Fedarko NS, Jain A, et al. Evaluation of osteopontin as biomarker for pancreatic adenocarcinoma. Cancer Epidemiol Biomarkers Prev, 2004, 13, 487-491.
19. Le QT, Sutphin PD, Raychaudhuri S, et al. Identification of osteopontin as a prognostic plasma marker for head and neck squamous cell carcinomas. Clin Cancer Res, 2003, 9, 59-67.
20. Singhal H, Bautista DS, Tonkin KS, et al. Elevated plasma osteopontin in metastatic breast cancer associated with increased tumor burden and decreased survival. Clin Cancer Res, 1997, 3,605-611.
21. Hotte S J, Winquist EW, Stitt L, et al. Plasma osteopontin: associations with survival and metastasis to bone in men with hormone-refractory prostate carcinoma. Cancer, 2002, 95, 506-512.
22. Das R, Mahabeleshwar GH, Kundu GC. Osteopontin induces AP-1-mediated secretion of urokinase-type plasminogen activator through c-Src-dependent epidermal growth factor receptor transactivation in breast cancer cells. J Biol Chem, 2004, 279:11051-11064.
23. Das R, Mahabeleshwar G.H, Kundu GC. Osteopontin stimulates cell motility and nuclear factor kappaB-mediated secretion of urokinase type plasminogen activator through phosphatidylinositol 3-kinase/Akt signaling pathways in breast cancer cells. J Biol Chem, 2003, 278: 28593-28606.
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2. Rittling SR, Chambers AF. Role ofosteopontin in turnout progression. Br J Cancer, 2004, 90 : 1877-1881.
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7. Yeatman TJ, Chamers AF. Osteopontin and colon cancer progression. Clin Cancer Res, 2003, 20, 85-90.
8. Das R, Mahabeleshwar GH, Kundu GC. Osteopontin induces AP-1-mediated secretion of urokinase-type plasminogen activator through c-Src-dependent epidermal growth factor receptor transactivation in breast cancer cells. J Biol Chem, 2004, 279:1051-11064.
9. Philip S, Bulbule A, Kundu GC. Osteopontin stimulates tumor growth and activation ofpromatrix metalloproteinase-2 through nuclear factor-kappa B-mediated induction of membrane type 1 matrix metalloproteinase in murine melanoma cells. J Biol Chem, 2001, 276: 44926-44935.
10. Jain S, Chakraborty G, Kundu GC. The crucial role of cyclooxygenase-2 in osteopontin-induced protein kinase C alpha/c-Src/IkappaB kinase alpha/beta-dependent prostate tumor progression and angiogenesis. Cancer Res, 2006,66: 6638-6648.
11. Cook AC, Chambers AF, Turley EA, et al. Osteopontin induction of hyaluronan synthase 2 expression promotes breast cancer malignancy. J Biol Chem, 2006, 281:24381-24389.
12. Gotoh M, Sakamoto M, Kanetaka K, et al. Overexpression of osteopontin in hepatocellular carcinoma. Pathol Int, 2002,52:19-24.
13. Pan HW, Ou YH, Peng SY, et al. Overexpression of osteopontin is associated with intrahepatic metastasis, early recurrence, and poorer prognosis of surgically resected hepatocellular carcinoma. Cancer, 2003, 98: 119-127.
14. Ye QH, Qin LX, Forgues M, et al. Predicting hepatitis B virus-positive metastatic hepatocellular carcinomas using gene expression profiling and supervised machine learning. Nat Med, 2003, 9: 416-423.
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