【作者】 李文昌；

【导师】 叶胜龙； Hong Zhang； 刘银坤；

【作者基本信息】 复旦大学 ， 肿瘤学， 2004， 博士

【摘要】 肝细胞性肝癌的术后复发和转移是导致其预后差的一个重要原因，而大部分的复发是由于侵袭相关的播散。因此，研究肝癌侵袭和转移的机制以及探索干预策略成为迫切需要。我所近期研究发现：伴随转移的原发性肝癌与其相应的转移灶的基因表达非常相似，提示有助于转移进展的基因变化在原发肿瘤阶段即已形成，因此研究与肝癌侵袭转移密切相关的根本分子机制非常重要。信号转换和转录激活因子3（signal transducer and activator of transcription 3,Stat3）调控由细胞因子以及生长因子受体信号传导通路引起的基因表达。而在人类的多种肿瘤中发现持续活化的Stat3。持续激活的Stat3信号途径通过与生长因子信号系统、细胞凋亡、血管形成等相互作用而导致肿瘤生成。Stat3作为抗肿瘤治疗靶点的价值已经在多种肿瘤细胞系中得到证实。虽然最近研究表明肝癌组织中存在显著的Stat3蛋白的上调或者磷酸化Stat3（Tyr705）过度表达，但是对其在肝癌发生、进展中的机制尚无深入阐释，亦未见到以其为靶点进行抗肝癌治疗的报道。反义寡核苷酸作为一种新的生物、基因治疗的手段，应用于多种疾病，尤其是恶性肿瘤的靶向干预治疗。抗肿瘤反义寡核苷酸治疗所针对的分子靶点包括与侵袭转移、细胞凋亡、多药耐药、血管形成、细胞内信号传导通路等相关的基因以及端粒酶等。至目前，已经有几种反义寡核苷酸药物已经进入临床试验。在第一代硫代反义寡核苷酸基础上研发的第二代——2’-MOE修饰的硫代间隙反义寡核苷酸既具有耐受核酸酶、激活RNase H的作用，又有增强的结合靶mRNA的活性和代谢稳定性，其更为有利的生化、药代动力学特性使之有望成为广泛应用于临床的反义寡核苷酸药物。因此，本课题旨在研究Stat3信号通路在肝癌侵袭转移过程中的作用及其作为抗肝癌反义治疗分子靶点价值；探讨2’-O-（2-甲氧乙烷基）（2’-MOE）修饰的反义Stat3寡核苷酸药物体外、体内干预肝癌生长、侵袭转移、诱导血管生成能力等的作用，并探讨其分子机制；从而为该药的进一步研究奠定基础，同时也为深入理解肝癌发生、侵袭转移的机制提供新的思路。第一部分2’-MOE修饰反义Stat3寡核苷酸对高侵袭性人肝癌细胞的作用及其分子机制研究本实验主旨在于研究2’-MOE修饰的反义Stat3寡核苷酸特异抑制高侵袋性人肝癌细胞系HCCLM3中Stat3表达的规律，观察其对该细胞系增殖、体外侵袭、诱导血管生成等潜能的作用，并利用cDNA微阵列技术探讨其相关的分子机制，为该药物的进一步研究奠定基础，同时为深入理解肝癌生成、进展的机制提供新的思路。1．反义Stat3寡核苷酸特异抑制HCCLM3细胞Stat3表达的规律：采用Lipofectamine^TM 2000阳离子脂质体转染试剂作为载体（寡核苷酸：脂质体=1：2（w／v）），以空白载体为对照，分别以2’-MOE修饰的反义Stat3寡核苷酸（Stat3 ASO,ISIS-113176-3）、随机序列寡核苷酸（BRC oligon.,ISIS-29848-11）转染HCCLM3细胞。分别应用RT-PCR、免疫细胞化学的方法检测转染细胞Stat3 mRNA以及Stat3蛋白、磷酸化Stat3（Try705）蛋白表达水平的变化。结果发现：Stat3 ASO以剂量依赖性、序列特异性的方式抑制HCCLM3细胞系Stat3 mRNA表达，其IC50剂量（24h）约为250nM。Stat3蛋白、磷酸化Stat3（Try705）蛋白的表达被显著抑制。2．反义Stat3寡核苷酸对细胞形态及超微结构的影响：用相差显微镜以及电子显微镜观察转染Stat3 ASO或BRC oligon．细胞的形态以及超微结构，结果发现：Stat3 ASO和阳离子脂质体形成直径约300nm的单层脂质体复合物，以胞吞方式进入细胞。Stat3 ASO能够特异地引起线粒体嵴减少。3．反义Stat3寡核苷酸对细胞增殖、成瘤性、细胞凋亡和细胞周期的影响及其可能分子机制：采用MTT法、裸鼠体内成瘤实验评价药物对细胞增殖、体内成瘤性的影响，流式细胞仪分析细胞周期和细胞凋亡。结果发现：Stat3 ASO以剂量依赖的方式特异抑制HCCLM3细胞的增殖，并且其抑制细胞增殖的效应在48h达到高峰；Stat3 ASO预处理（250nM，24h）的细胞在裸鼠体内的成瘤性减弱以及增长缓慢。250nM Stat3 ASO处理细胞24h，引起细胞周期S／G2-M期阻滞、诱导明显的细胞凋亡（43.6±1.83％）。基因芯片发现Stat3 ASO下调了细胞周期及DNA损伤修复相关的关键基因BRCA1、BRCA2、cyclin D1、p16ink4、CDC25a、cdk4，p21Wafl等，下调了与凋亡有关的基因Apaf-1、bax、bcl-2、bcl-x、survivin等，并上调Fas（Apo-1）的表达。多基因的变化导致其上述效应。4．反义Stat3寡核苷酸对细胞体外侵袭能力的影响及其可能分子机制：Transwell体外侵袭实验测定发现；250nM Stat3 ASO处理细胞24h能够特异抑制细胞体外侵袭活性。明胶酶谱法测定显示：转染细胞MMP2、MMP9的分泌水平显著降低，Stat3 ASO特异下调HCCLM3细胞分泌MMP2、MMP9，尤其是MMP2。基因芯片结果显示：Stat3 ASO下调与侵袭转移相关的基因MMP-2、MMP-9、KISS1、Nm23-E4等，以及与粘附相关的基因Integrinα5、Integrinα3、CD44、MUC-18、Integrinβ3；并上调TIMP1。这些基因综合的影响结果导致肝癌细胞体外侵袭能力的下降。5．反义Stat3寡核苷酸对细胞诱导血管生成能力的影响及其可能分子机制：裸鼠皮内肿瘤血管形成试验发现：Stat3 ASO显著抑制了HCCLM3诱导血管生成的能力；RT-PCR和ELISA的方法显示：VEGF121；VEGF165；VEGF189三种VEGF同源体的表达显著下调。基因芯片进一步发现：与血管形成相关的基因如ANGPT2、EGFR、FGF2、IFNA1、IGF-1、PDGFα、thrombospondin2、VEGF等显著下调。另外，akt-1、erb-2、MEK1、β-catenin、c-fos等基因的下调表达可能参与了上述多个过程。综上所述，2’-MOE修饰的反义Stat3寡核苷酸特异抑制Stat3的表达，并影响多基因表达谱的改变，进而有效抑制了肝癌的生长、侵袭、血管生成能力。说明反义Stat3寡核苷酸药物是有潜力的抗肝癌治疗的药物。也从另外角度提示Stat3信号通路可能通过调控多种肿瘤发生相关基因而影响肝癌的进展。因而为肝癌分子机制的研究提供了新的思路。第二部分2’-MOE修饰反义Stat3寡核苷酸抑制肝癌生长和侵袭转移的体内研究本部分研究目的在于探讨体内应用2’-MOE修饰的反义Stat3寡核苷酸对有高侵袭潜能肝癌的生长、侵袭转移的作用，评价其作为抗肝癌治疗药物的应用价值。建立90只裸鼠原位种植HCCLM3肝癌模型，并随机平均分为两大组。各大组随机分为三实验组（n=15），分别设为：生理盐水（NS）对照组（G1）、随机序列寡核苷酸（BRC oligon.）对照组（G2）、反义Stat3寡核苷酸（Stat3 ASO,ISIS-113176-3）治疗组（G3）。方案一（E1）：其中一大组裸鼠于原位种植肿瘤后第2天开始治疗，E1G1组NS 0.2ml i.p.，E1G2组50mg／kg BRC oligon.ip.，E1G3组50mg/kg Stat3 ASO i.p.，均隔两天注射一次；连续注射9次后，改为隔一天注射一次，注射3次；方案二（E2）：另一大组裸鼠于种植肿瘤后第10天开始治疗，E2G1组NS 0.2ml i.p.，E2G2组50mg／kg BRC oligon.ip.，E2G3组50mg/kg Stat3 ASO i.p.，均隔一天注射一次，注射12次。最后一次给药后第三天（即第35天时），各组随机选择9只裸鼠解剖观察肝癌生长和腹腔转移情况；免疫组化检测Stat3表达；抗小鼠CD34抗体标记肝癌血管内皮细胞，计数微血管密度（MVD）；采用连续切片法检测肺转移灶数目；ELISA法测定裸鼠血清中VEGF和bFGF的含量；常规检验血细胞分类计数以及转氨酶（AST、ALT）等。各组中另6只裸鼠继续饲养以观察生存时间。我们获得了至少四方面有意义的结果：1．体内应用反义Star3寡核苷酸有效抑制肝癌的生长、侵袭转移，延长荷瘤鼠生存期：两种用药方案，反义Stat3寡核苷酸均能够特异地抑制Stat3蛋白的表达，并有效抑制肝癌的生长、局部转移（肝内、腹腔、膈肌等）和肺转移。方案一中反义Stat3寡核苷酸治疗组（E1G3）瘤重为0.93±0.12g，明显低于生理盐水对照组（E1G1）1.73±0.17g（P＜0.01）和随机序列寡核苷酸组（E1G2）1.67±0.27g（P＜0.01）。E1G3组未见肺转移，而E1G1组和E1G2组肺转移率分别为100％和89％（P＜0.001）。E1G3组荷瘤裸鼠的生存期显著长于E1G1组（101±14.6天vs 49.0±4.9天，P＜0.001）和E1G2组（101±14.6天vs 52.7±6.7天，P＜0.001）。方案二中反义Stat3寡核苷酸治疗组（E2G3）瘤重为1.18±0.11g，也明显低于生理盐水对照组（E2G1）1.89±0.24g（P＜0.01）和随机序列寡核苷酸组（E2G2）1.77±0.39g（P＜0.01）。E2G3组肺转移率为22％，而E2G1组、E2G2组的肺转移率皆为100％（P＜0.01）。E2G3组荷瘤裸鼠的生存期亦显著长于E2G1组（82.3±11.1天vs 46.2±4.3天，P＜0.001）和E2G2组（82.3±11.1天vs 50.2±6.2天，P＜0.001）。上述结果表明2’-MOE修饰反义Stat3寡核苷酸体内应用具有可观的抗肝癌生长、侵袭转移的治疗价值。2．体内应用反义Stat3寡核苷酸有效抑制肝癌血管生成：两种治疗方案中，反义Stat3寡核苷酸均能有效抑制肝癌血管生成，E1G3组微血管密度显著低于E1G1组（3.3±1.5 vs．15.2±3.4；P＜0.01）和E1G2组（3.3±1.5 vs．13.8±4.0；P＜0.01）；E2G3组微血管密度也明显低于E2G1组（4.0±1.8vs．16.8±1.7：P＜0.01）和E2G2组（4.0±1.8 vs．14.5±4.0；P＜0.01）。ELISA检测结果显示：反义Stat3寡核苷酸明显抑制了荷瘤裸鼠血清中VEGF和bFGF的表达水平（P＜0.01）。上述结果表明：通过下调VEGF和bFGF等促血管生成因子的表达而抑制血管生成可能是反义Stat3寡核苷酸抑制肝癌在裸鼠体内生长和转移的机制之一。3．肿瘤负荷影响反义Stat3寡核苷酸的疗效：早期给予反义Stat3寡核苷酸治疗（E1G3组）比后期给予反义Stat3寡核苷酸治疗（E2G3组）更为有效地抑制肝癌生长（瘤重0.93±0.12g vs．1.18±0.11g；P＜0.05）、肺转移（0／9 vs 2／9；P＜0.05）、以及延长荷瘤鼠的生存期（101±14.6天vs．82.3±11.1天；P＜0.05），但两组在Stat3蛋白表达、血清VEGF、bFGF含量和微血管密度等方面未见明显差别。4．体内应用反义Stat3寡核苷酸对荷瘤裸鼠的造血功能、肝脏功能无明显影响：综上所述，2’-MOE修饰的反义Stat3寡核苷酸体内应用展示其抗肝癌生长、侵袭、血管形成的治疗价值及其能被荷瘤宿主良好耐受的特性；首次揭示肿瘤负荷影响反义寡核苷酸抗肿瘤的疗效；证实Stat3信号通路在肝癌生成、进展中有重要作用，为深入研究肝癌发生、侵袭转移机制提供新的思路。更多还原

【Abstract】 Hepatocellular carcinoma （HCC） mostly warrants a very poor prognosis caused by the recurrence and metastasis after resection. The majority of recurrence after resection occurs due to invasion-related spreading. Thus, it is imperative to investigate the controlling mechanism of HCC metastasis and invasiveness and to explore the effective therapeutic strategies. The recent study in our institute indicates that the expression signature of primary HCC with accompanying metastasis is very similar to that of their corresponding metastases, implying that genes functioning for metastatic progression are initiated in the primary tumors. So, it is important to understand the fundamental molecular mechanisms involved in invasiveness and metastasis of HCC. The signal transducer and activator of transcription 3 （Stat3） regulates gene expression in response to the signaling from cytokine and growth factor receptors. However, Stat3 protein is constitutively activated in a wide variety of human tumors with high frequency. Persistent signaling of Stat3 has been demonstrated to contribute to oncogenesis through their intimate connection to the signaling relating to growth, apoptosis, or angiogenesis. Stat3 as a novel drug target for therapeutic intervention of human cancers has been shown to be valuable. Recent studies have indicated that Stat3 protein or phospho-Stat3 （Tyr705） protein is overexpressed in HCC, however, it is mostly unknown at present how the signaling of Stat3 is implicated in HCC carcinogenesis and development and how the efficacy of treatment targeting Stat3 on HCC. As a novel gene-based therapeutic approach , antisense oligonucleotides have been used as the targeted molecular treatment of various diseases, especially malignant tumors. Antisense approaches to cancer therapy have been explored for a myriad of targets, including gene products involved in tumor invasiveness, the anti-apoptosis cascade, multidrug resistance and telomerase. At present, several antisense oligonucleotides have entered clinical use or clinical trials. On the basis of the first generation phosphorothioate antisense oligonucleotide, the second generation 2’-O-（2-methoxyethyl） （2’-MOE）-modified phosphorothioate gapmer antisense oligonucleotide has been developed which has increased both resistance to nuclease degradation, activation of RNase H, and binding affinity for target mRNA , metabolic stability. The favorable biochemical and pharmacokinetic characteristics of 2’-MOE-modified antisense oligonucleotide make it as a promising therapeutic drug entering clinical use. In the present study, we have investigated the role of the signaling of Stat3 in HCC growth, invasiveness, and metastastasis; and evaluated the efficacy of 2’-O-（2-methoxyethyl）-modified antisense Stat3 oligonucleotides on growth, invasiveness of human high metastatic hepatocellular carcinoma in vitro and in vivo; and studied the molecular mechanisms. Our study provides a basis for further evaluation of 2’-MOE modified antisense Stat3 oligonucleotides as a anticancer drug; and provides a novel pathway to understand the mechanism of HCC invasiveness and metastasis.PartⅠEfficacy of 2’-O-（2-Methoxyethyl）-Modified Antisense Stat3Oligonucleotides on Human Hepatocellular Carcinoma Cells with High Metastatic Potential and Further Study of the Molecular MechanismsThe objective of the present study was to explore how the 2’-O-（2-methoxyethyl） modified antisense Stat3 oligonucleotides specially inhibited the expression of Stat3 in HCCLM3 cells with high metastatic potential; and to investigate the efficacy of the drug on the growth, invasiveness, and angiogenesis of the cells in vitro; and to analyze the molecular changes caused by this drug using cDNA array; so as to provide a basis for further study of this drug and to provide a novel pathway to understand the mechanism of HCC invasiveness and metastasis.1．2’-MOE Stat3 ASO significantly inhibited the expression of Stat3 in HCCLM3 cellsOligonucleotides were delivered to HCCLM3 cells in the form of complexes with Lipofectamine^TM2000 （the ratio of oligo:lipo. was 1: 2（w/v））. HCCLM3 cells were treated with 2’-MOE Stat3 ASO （ISIS-113176-3） , base-randomized control oligonucleotides （BRC oligon. ISIS-29848-11） , or Lipofectamine alone as a control. The Stat3 mRNA and Stat3 protein and phospho-Stat3 （Tyr705） protein were assayed by RT-PCR or immunocytochemistry, respectively. The results indicated that the expression of Stat3 mRNA in HCCLM3 was inhibited by 2’-MOE Stat3 ASO in a concentration-dependent and sequence-specific fashion with with an IC50 of about 250 nM （51% inhibiton rate）. The expression of Stat3 protein or phospho-Stat3 （Tyr705） protein was also significantly downregulated by Stat3 ASO.2．The effect of 2’-MOE Stat3 ASO on cell morphology and ultrastructure of HCCLM3 cellsThe cell morphology and ultrastructure of HCCLM3 cells was observed using reverse-microscope and electronic microscope respectively. The complex of liposome and oligonucleotides was entered cells in a form of monolayer liposome complex 300nm in diameter. The number mitochondrial critae in HCCLM3 cells transfected with Stat3 ASO was reduced compared with cells treated with BRC oligon.3．2’-MOE Stat3 ASO impeded cell growth and tumorigenicity, induced cell apoptosis and cell cycle S /G2-M phase ArrestThe in vitro growth-inhibitory effect of the 2’-MOE Stat3 ASO on HCCLM3 cells was evaluated using MTT assay. The ability of umorigenicity was detected by in vivo tumorigenecity assay. Apoptosis and cell cycle analysis were performed by flow cytometer. The results showed that Stat3 ASO reduced HCCLM3 cell growth in a sequence-specific and dose dependent manner with a peak of the inhibition at 48h. The tumors induced by HCCLM3 cells pretreated with AS-Stat3 odn growed slower than those induced by cells pretreated with BRC oligon. or lipofectamine only. AS-Stat3 odn resulted in enhanced blockade of cell cycle progression at the S/G2-M checkpoint and induced significant cell apoptosis. CDNA array analysis indicated that key genes associated with cell cycle or DNA damage respair such as BRCA1、BRCA2、cyclinD1、p16ink4、CDC25a、cdk4, p21Waf1 etc. and genes on apoptosis such as Apaf-1、bax、bcl-2、bcl-x、survivin etc. were significantly down-regulated after inhibition of Stat3 by Stat3 ASO. The Fas （Apo-1） gene was upregulated. The changes of multiple genes described above resulted in the efficacy we observed.4．2’-MOE Stat3 ASO inhibited cell invasion of HCCLM3 cellsThe invasive ability of cells was evaluated in 24-well matrigel precoated filters Transwell chambers. Furthermore, we analyzed the secretion of matrix metalloproteinase-2 and-9 （MMP2 and MMP9） using zymogram assay. The results showed that the treatment of 250nM Stat3 ASO （24h） resulted in decreased invasive ability of HCCLM3 cells. The Stat3 ASO significantly reduced the secretion of MMP2 and MMP9, especially MMP2．CDNA array analysis indicated that genes on invasion such as MMP-2, MMP-9, KISS1, Nm23-E4, etc. and genes on adhesion such as Integrinα5, Integrinα3, CD44, MUC-18, Integrinβ3, etc.) were significantly down-regulated by Stat3 ASO. The TIMP1 gene was upregulated by Stat3 ASO. The changes of these genes might be involved in the mechanisms of inhibiting invasiveness.5．2’-MOE Stat3 ASO inhibited angiogenesis induced by HCCLM3 cellsThe intradermal angiogenesis assay in nude mice indicated that angiogenesis induced by cells treated with Stat3 ASO was significantly reduced ,compared with BRC oligon.. The results from RT-PCR and ELISA showed that three isoforms of VEGF （VEGF121, VEGF165, VEGF189） were downregulated by Stat3 ASO treatment. CDNA array analysis demonstrated that genes related to angiogenesis such as ANGPT2, EGFR, FGF2, IFNA1, IGF-1, PDGFα, thrombospondin2, VEGF, etc. were downregulated by inhibiton of Stat3． In addition, some key genes related to signal transduction pathway such as akt-1, erb-2, MEK1,β-catenin, c-fos etc. were also downregulated by Stat3 ASO. These genes were perhaps involved in multiple pathways described above.To sum up, 2’-MOE modified Stat3 antisense oligonucleotides significantly inhibited the expression of Stat3 , which leaded to the disruption of Stat3 signaling pathway. As a result, the growth, invasion, and angiogenesis of HCC were impeded obviously. We can conclude that 2’-MOE modified Stat3 antisense oligonucleotide is an anticancer drug with therapeutic potential. On the other hand, Stat3 signling pathway plays an important role in the development of HCC.PartⅡInhibition of growth and metastasis of human hepatocellular carcinoma growing in nude mice by 2’-O-（2-Methoxyethyl）-Modified Antisense Stat3 OligonucleotidesThe purpose of the present study was to investigate the efficacy of 2’-O-（2-methoxyethyl）-modified antisense Stat3 oligonucleotides on growth, invasiveness of human high metastatic hepatocellular carcinoma by using in vivo nude mice xenograft models, and to evaluate it as an anti-cancer therapeutic drug.A highly metastatic xenograft model of human hepatocellular carcinoma （HCC） was established in nude mice via orthotopic implantation of HCCLM3 cells with a highly metastatic potential. 90 mice with transplanted tumours were randomly assigned to two large groups .Mice in each large group were then randomized into three experimental groups （n=15） as follows: （G1） i.p. injections of physiological physiological saline as a control; （G2） i.p. injections of 50mg/kg base-randomized control oligonucleotides （BRC oligon.）; （G3） i.p. injections of 50mg/kg 2’-MOE modified antisense Stat3 oligonucleotides （Stat3 ASO, ISIS-113176-3） . After randomization, mice in two large group respectively received experimental antisense therapy in different schemes. In a first in vivo experiment （E1） , treatment of mice in each experimental group was started at the second day after tumor implantation. 50mg/kg of Stat3 ASO （group E1G3） , or BRC oligon. （group E1G2） , or 0．2ml of physiological saline （group E1G1） was injected i.p. once every three days for 9 times, and then injected i.p. once every two days for another 3 times. In a second in vivo experiment （E2） , treatment of mice in each experimental group was started at the tenth day after tumor implantation. 50mg/kg of Stat3 ASO （group E2G3） , or BRC oligon. （group E2G2） , or 0．2ml of physiological saline （group E2G1） was injected i.p. once every two days for 12 times. Three days after the last injection （i.e. 35 days after tumor implantation）, randomly selected mice from each experimental group （n=9） were humanely sacrificed to observe the growth and matastasis of HCCLM3 tumors ,and tumors and lungs were harvested. Human Stat3 and mouse CD34 were assessed by immunohistochemistry and positive nuclei of Stat3 and microvessel density （MVD） labeled by CD34 were measured. Pulmonary metastases were detected by observing H.E. stained sequential sections of paraffin-embedded lung tissues. Blood samples were collected to allow for routine blood analysis and to determine AST, ALT in serum. The determination of VEGF, bFGF in serum was performed by ELISA. Another 6 mice in each group were maintained to evaluate life span.In the present study, we have demonstrated at least four significant results:1．Treatment with 2’-MOE modified Stat3 ASO effectively inhibited growth and metastasis of HCCLM3 tumors orthotopically implanted in nude mice, and obviously prolonged life span of mice with tumors.In two experiments, the expression of Stat3 protein in tumors was inhibited significantly and specifically by Stat3 ASO. As a result, the growth, local metastasis （intrahepatic, intra-celiac, diaphragmatic,etc.） ,and lung metastasis were impeded significantly. In the first experiment, treatment with Stat3 ASO （group E1G3） significantly decreased the mean tumor weight compared with control mice （group E1G1, E1G2） （0．93±0．I2g, 1．73±0．17g, 1．67±0．27g, respectively; P<0．01） . Lung metastasis was not found in group E1G3, however, incidence of lung metastasis in control group E1G1 and E1G2 was 100% （P<0．001） , 89% （P＜0．001） , respectively. Mice in group E1G3 obtained prolonged life span compared with control group E1G1（101±14．6 days vs 49．0±4．9 days, P<0．001） , or with group E1G2 （101±14．6 days vs 52．7±6．7 days, P<0．001） . In the second experiment, treatment with Stat3 ASO （group E2G3） also significantly decreased the mean tumor weight compared with control mice （group E2G1, E2G2） （1．18±0．11g, 1．89±0．24g, 1．77±0．39g respectively; P<0．01） . Lung metastasis in group E2G3 was also inhibited significantly compared with control E2G1 （22% vs 100% , P<0．01） or E2G2 （22% vs 100% , P<0．01） . Mice in group E2G3 obtained prolonged life span compared with control group E2G1（82．3±11．1days vs 46．2±4．3 days, P<0．001） , or with group E2G2 （82．3±11．1days vs 50．2±6．2 days, P<0．001） .2．Treatment with 2’-MOE modified Stat3 ASO effectively inhibited angiogenesis in HCCLM3 tumors.In both experiments, treatment with Stat3 ASO significantly inhibited microvessel density （MVD） compared control mice. MVD in group E1G3 was obviously decreased compared with control group E1G1 （3．3±1．5 vs.15．2±3．4; P<0．01） , or with group E1G2 （3．3±1．5 vs.13．8±4．0; P＜0．01） . MVD in group E2G3 was also obviously decreased compared with control group E2G1 （4．0±1．8 vs. 16．8±1．7; P<0．01） , or with group E2G2 （4．0±1．8 vs.14．5±4．0; P<0．01） . The results of ELISA assay showed that treatment of Stat3 ASO significantly decreased the secretion of VEGF and bFGF. These results indicated that downregulation of VEGF and bFGF is one of the mechanisms that Stat3 ASO inhibited the growth and metastasis of HCCLM3 tumor in vivo.3．Tumorous burden impacted on the efficacy of Stat3 ASO.Treatment with Stat3 ASO started at the second day after tumor implantation （group E1G3） showed more efficacy than treatment started at the tenth day after implantation （group E2G3） on tumor weight （0．93±0．12g vs. 1．18±0．11g; P<0．05） , incidence of lung metastasis （0 vs 22%; P<0．05 ）, life span （101±14．6 days vs. 82．3±11．1days; P<0．05） . However, the difference between two groups had no statistic significance in the expression of Stat3, serum VEGF/bFGF level and MVD.4．Treatment with 2’-MOE modified Stat3 ASO had no obvious hepatic or blood toxicity.Thus, disruption of Stat3 by using antisense approach may serve as a novel therapeutic strategy for hepatocellular carcinoma. Our results provide a basis for further evaluation of 2’-MOE modified antisense Stat3 oligonucleotides as an anticancer drug.更多还原