摘要
我国原发性肝癌病死率居所有恶性肿瘤的第二位,占全部恶性肿瘤死亡人数的18.8%。乙型肝炎、肝炎后肝硬化则是我国肝癌发病率和病死率居高不下的主要原因。虽然手术切除仍是可能获得治愈的主要方法,但根治性切除术后复发率仍然相当高,还有更多的肝癌患者因肿瘤转移而失去根治的机会。这是肝癌患者临床治疗失败的主要原因,也是提高患者生存率和改善预后的重要障碍。因此探讨肝癌发生发展和转移复发的相关因素及分子机理,寻找肝癌早期诊断、预测转移的生物标记和干预治疗的靶分子,对于这种恶性肿瘤的诊断和治疗具有重要的理论指导和临床应用意义。
Survivin基因由效应蛋白EPR-1(effector cell protease receptor-1)cDNA在人类基因库杂交筛选分离出来,全长14,700 bp,定位于17q25,编码产生一个由142个氨基酸组成、相对分子质量为16,500的蛋白质。Survivin的主要生物学功能是抗凋亡,是目前已知作用最强的凋亡抑制蛋白。目前,越来越多的学者发现Survivin在肿瘤细胞中表达,特别是在高转移潜能的肿瘤细胞中表达。有学者报道Survivin在转移的肝癌患者样本中表达的阳性率显著高于无转移患者,Survivin表达可能与原发性肝癌的肝内转移、门静脉癌栓等恶性生物学行为有关,但该方面研究较少,而且具体作用机制如何还不明确。
本课题旨在通过对比和分析不同转移潜能人肝癌细胞系Survivin表达差异及肝癌组织有和无肝内转移病例中Survivin表达差异,研究Survivin与肝癌侵袭转移之间的关系;通过构建Survivin表达载体pEGFP-C1/survivin重组质粒,研究Survivin过表达后对肝癌细胞侵袭、转移恶性表型的影响;通过构建Survivin RNAi真核表达载体pGPU6/GFP/Neo-shRNA,探索下调Survivin基因表达对肝癌侵袭、转移的治疗效果,为肝癌转移的基因治疗提供新的靶点;并初步研究Survivin促进肝癌侵袭转移的作用机制。
第一部分:Survivin在不同转移潜能肝癌细胞系及肝癌组织中的表达
本部分实验目的是研究不同转移潜能人肝癌细胞系及肝癌组织中Survivin表达情况,分析Survivin表达与肝癌侵袭、转移之间的关系。应用RT-PCR和WesternBlot方法检测5种不同转移潜能人肝癌细胞系SMMC-7721、MHCC97-L、MHCC97-H、HCCLM3和HCCLM6细胞Survivin表达情况。收集35例原发性肝癌组织标本及相应的癌旁组织,其中包括20例无肝内转移病例及15例存在肝内转移的病例,RT-PCR和Western Blot方法检测Survivin表达情况。RT-PCR结果表明,具有相同遗传背景的转移潜能肝癌细胞系随侵袭转移潜能升高Survivin表达依次升高(P＜0.05),HCCLM6细胞Survivin表达水平最高,Western Blot结果与RT-PCR结果一致。肝癌组织Survivin mRNA表达均为阳性,而癌旁组织无一例阳性表达。Survivin mRNA的表达水平在肝内转移组(1.082±0.213)显著高于无肝内转移组(0.799±0.224,P＜0.05)。Western Blot的结果与RT-PCR结果一致。肝内转移组Survivin蛋白表达(0.892±0.198)显著高于无肝内转移组(0.342±0.237,P＜0.05)。以上结果表明,Survivin在原发性肝癌中表达,且与侵袭转移等肿瘤的恶性生物学行为有关,Survivin基因高表达可作为判断HCC侵袭性和进展的一个潜在重要指标。
第二部分:Survivin过表达对肝癌细胞恶性表型的影响
本部分实验目的是研究Survivin过表达后对肝癌细胞恶性表型的影响。首先构建Survivin表达质粒pEGFP-C1/survivin,将其转染低侵袭性无转移潜能人肝癌细胞SMMC-7721,以pEGFP-C1空质粒转染做对照,G418筛选获得稳定过表达Survivin细胞株C1和稳定转染空质粒细胞株C2,用Realtime PCR和Western Blot方法检测Survivin表达,体外MTT、黏附、运动、侵袭实验观察Survivin过表达后细胞恶性表型的变化。制备18只SMMC-7721细胞裸鼠肝脏原位接种模型,随机平均分成pEGFP-C1/survivin质粒组,pEGFP-C1空质粒对照组和PBS组。在肝脏原位接种肿瘤后第2天开始给药,腹腔注射质粒50gg/只,每周2次,连续给药6周后处死,解剖观察肝癌生长、转移情况,RT-PCR、Western Blot检测肝癌组织中Survivin和OPN表达,肺组织切片H.E.染色观察肺转移情况。肿瘤转移相关基因芯片比较C1和C2细胞株基因表达差异。用Realtime PCR验证有差异表达H-ras基因。并检测OPN基因、蛋白表达情况。结果表明,pEGFP-C1/survivin质粒经测序鉴定构建正确,将其转染SMMC-7721细胞,Survivin表达水平明显升高。体外试验显示C1细胞黏附、体外运动、侵袭能力明显高于C2细胞(细胞黏附率65.33%±7.24%vs 45.34%±5.52%,运动试验穿膜细胞数15.3±2.34 vs 5.3±2.53,侵袭试验透膜细胞数7.3±1.34 vs 4.3±1.25,P均＜0.05),MTT结果显示C1细胞24h、48h和72h三个时间点检测的OD值明显高于C2细胞检测值(P＜0.05)。裸鼠实验结果显示,pEGFP-C1/survivin质粒组肿瘤瘤重(2.72g±0.16g)明显重于pEGFP-C1空质粒对照组(1.79g±0.08g,P＜0.05)和PBS组(1.75g±0.07g,P＜0.05)。各组肝癌体积的差异趋势与瘤重一致,pEGFP-C1/survivin质粒组体积(1062.1mm~3+376.0mm~3)明显大于pEGFP-C1空质粒对照组(409.7mm~3±380.3mm~3,P＜0.01)和PBS组(460.8mm~3±32.3mm~3,P＜0.01)。pEGFP-C1/survivin质粒组6只裸鼠中有5只发生肺转移,pEGFP-C1空质粒对照组和PBS组均无肺转移发生,且pEGFP-C1/survivin质粒组3只发生肝内播散,而pEGFP-C1空质粒对照组和PBS组裸鼠均无肝内播散。RT-PCR及Western Blot结果显示pEGFP-C1/survivin质粒组Survivin和OPN表达水平明显高于pEGFP-C1空质粒对照组和PBS组。基因芯片比较发现稳定高表达Survivin后,有16个肿瘤转移相关基因表达上调(H-ras、CTNNA1、CTNNB1、IL8RB、MMP3、ITGB3等),2个肿瘤转移相关基因表达下调(TCF20和TP53)。Realtime PCR和ELISA结果显示C1细胞H-ras和OPN表达水平明显高于C2细胞。以上结果表明,Survivin基因表达上调后可促进肝癌细胞SMMC-7721的恶性侵袭转移表型。Survivin高表达可引起多个肿瘤转移相关基因表达水平改变,其中包括与肝癌侵袭、转移密切相关的H-ras和OPN基因。
第三部分:Survivin基因RNAi体外及体内研究
本部分实验进一步观察Survivin对肝癌细胞侵袭转移能力的影响,通过靶向Survivin RNA干扰抑制Survivin表达,观察高转移人肝癌细胞系HCCLM6细胞生长、黏附、侵袭能力以及体内肿瘤生长及转移行为的改变,探索靶向Survivin基因RNAi对肝癌侵袭、转移的治疗效果。首先设计、合成Survivin siRNA,瞬时转染HCCLM6细胞,Realtime PCR和Western Blot检测干扰效果。筛选出干扰效率较高靶点用于合成shRNA,构建pGPU6/GFP/Neo-shRNA干扰质粒。重组质粒瞬时转染HCCLM6细胞,Realtime PCR和Western Blot验证干扰效果,筛选出干扰效率较高的shRNA重组质粒进行后续体内、体外实验。应用MTT、黏附实验、细胞运动、侵袭实验观察细胞增殖、黏附、运动、侵袭能力的改变。Realtime PCR及ELlSA方法检测瞬时转染shRNA干扰质粒后细胞H-ras和OPN表达。裸鼠实验观察腹腔注射Survivin干扰质粒对肝癌生长、转移和裸鼠生存期的影响。RT-PCR和Western Blot检测裸鼠肝癌组织Survivin和OPN表达水平。结果表明,针对Survivin基因设计的siRNA均有一定程度的干扰作用,Realtime PCR显示其中siRNA-166、siRNA-358的干扰作用较强,可使靶基因分别下调80%和70%,WesternBlot结果与Realtime PCR结果一致,siRNA-166和siRNA-358可使Survivin蛋白分别下调72%和65%。分别构建siRNA-166和siRNA-358两种pGPU6/GFP/Neo-shRNA重组质粒,并分别命名为shRNA1和shRNA2,经测序鉴定插入位点正确。Realtime PCR检测两种重组质粒干扰效果,结果示shRNA1和shRNA2对Survivin mRNA抑制率分别为78%和65%,蛋白抑制率达75%和68%,故选择shRNA1进行后续实验。shRNA1转染HCCLM6后,体外增殖能力明显降低,黏附率明显低于阴性对照质粒转染细胞,两者差异有显著性(43.28%±0.85%vs55.09%±1.45%,P＜0.01);细胞运动、侵袭能力明显降低,与阴性对照质粒相比两者差异有显著性(侵袭试验透膜细胞数10.5±1.29 vs 20.5±1.29,P＜0.0l,运动试验穿膜细胞数15.8±0.96 vs 23.5±1.29,P＜0.01)。Realtime PCR结果显示细胞瞬时转染shRNA1干扰质粒后,细胞H-ras和OPN mRNA表达降低,细胞上清OPN蛋白分泌量明显减少(P＜0.0])。裸鼠实验表明,shRNA1质粒组肿瘤瘤重(1.43g±0.20g)低于阴性对照质粒组(2.]9g±0.37g,P＜0.01)和PBS组(2.34g±0.55g,P＜0.01)。shRNA1质粒组肿瘤体积(679.22mm~3±328.72mm~3)小于阴性对照质粒组(1397.03mm~3±374.49mm~3,P＜0.01)和PBS组(1636.50mm~3±508.80mm~3,P＜0.01)。阴性对照质粒组和PBS组6只裸鼠都发生了肺转移,而shRNA1质粒组只有2只裸鼠发生肺转移。shRNA1质粒组荷瘤裸鼠的生存期为70.7天±1.97天,与阴性对照质粒组(40.8天±2.32天)和PBS组(37.5天±4.28天)相比差异显著(P＜0.01)。RT-PCR及Western Blot结果显示shRNA1质粒组Survivin和OPN表达水平明显低于阴性对照质粒组和PBS组。以上结果表明,Survivin基因可通过促进肝癌细胞增殖,增强细胞黏附力、细胞运动、侵袭能力参与原发性肝癌的生长和转移,Survivin基因下调后肝癌细胞侵袭潜能下降,裸鼠肝癌生长抑制,转移能力降低,故认为Survivin是潜在的治疗原发性肝癌侵袭、转移的靶标。
第四部分:Survivin促进肝癌细胞恶性表型机制的初步研究
第二、三部分研究结果表明,Survivin基因过表达后,OPN和H-ras基因表达上调;而当干扰Survivin基因表达时,OPN和H-ras基因表达下调。本部分通过研究三者之间的关系,以初步探讨Survivin促进肝癌侵袭转移的作用机制。首先构建Survivin表达质粒pcDNA3.1(-)/survivin和Survivin RNA干扰质粒pGPU6/shRNA,并分别瞬时转染细胞检测Survivin表达变化。构建含有OPN基因启动子的荧光素酶报告质粒pOPNGL3并鉴定。设计并化学合成H-ras siRNA序列,瞬时转染HCCLM6细胞观察千扰效果,筛选出干扰效果较好的靶点用于后续实验。将pcDNA3.1(-)/survivin和pOPNGL3共转染SMMC-7721细胞,通过检测荧光素酶活性反映OPN基因启动子活性,观察OPN基因启动子活性的变化。将pGPU6/shRNA和pOPNGL3共转染HCCLM6细胞,观察OPN基因启动子活性的变化。将H-ras siRNA和pOPNGL3共转染HCCLM6细胞,检测OPN基因启动子活性。将pcDNA3.1(-)/survivin、H-ras siRNA和pOPNGL3共转染HCCLM6细胞,与共转染pcDNA3.1(-)/survivin、siRNA-NC、pOPNGL3细胞(对照组1)和共转染pcDNA3.1(-)、siRNA-NC、pOPNGL3细胞(对照组2)对比,观察OPN基因启动子活性差异。结果表明:通过测序鉴定pcDNA3.1(-)/survivin和pGPU6/shRNA重组质粒构建正确。pcDNA3.1(-)/survivin重组质粒瞬时转染SMMC-7721细胞后,Survivin mRNA表达水平升高,为SMMC-7721细胞2.173倍,高于空质粒转染细胞(相对SMMC-7721细胞1.035倍)。干扰质粒pGPU6/shRNA瞬时转染HCCLM6细胞后,Survivin mRNA表达水平明显低于空质粒转染细胞,抑制效率达70.5%。测序鉴定OPN基因启动子的荧光素酶报告质粒pOPNGL3构建正确。4组H-rassiRNA分别瞬时转染HCCLM6细胞进行Realtime PCR检测,结果发现v-H-ras-121、v-H-ras-282、v-H-ras-305、v-H-ras-489干扰效率分别是51%、75.3%、65.6%、34.5%,故选择v-H-ras-282进行后续试验。Survivin过表达后,SMMC-7721细胞OPN基因启动子活性升高了187.76%。Survivin基因干扰后,HCCLM6细胞OPN启动子活性降低了61.3%。H-ras基因干扰后,HCCLM6细胞OPN启动子活性降低了59.5%。pcDNA3.1(-)/survivin、H-ras siRNA共转染HCCLM6细胞,三组RLU(相对发光值)分别是16.31±0.67(×10~4)、37.69±1.34(×10~4)和15.86±0.32(×10~4),实验组与对照组1相比,荧光素酶相对活性明显低于对照组1(P＜0.01),而与对照组2相比,没有明显差异(P＞0.05),说明Survivin过表达对OPN启动子的激活作用,可以被H-ras表达下调所阻断。以上结果表明,Survivin过表达可以促进OPN基因启动子活性;H-ras基因可以通过调控OPN启动子活性,调控OPN基因表达;Survivin促进OPN启动子活性的作用可以被H-ras RNAi阻断。故认为Survivin、H-ras和OPN三者之间可能存在密切的联系,Survivin可能是通过促进H-ras基因表达激活OPN基因转录,进而使其表达上调,发挥促进肝癌侵袭转移的作用。
结论
1.Survivin表达和肝癌细胞侵袭转移能力相关:肝癌组织中Survivin表达与肝内转移有关。
2.Survivin能促进肝癌细胞系SMMC-7721侵袭转移恶性表型。Survivin高表达可引起SMMC-7721细胞H-ras、OPN等多个肿瘤转移相关基因表达改变。
3.RNA干扰下调Survivin表达后,肝癌细胞HCCLM6增殖、黏附、侵袭能力下降,并引起裸鼠体内肿瘤生长抑制及转移能力下降,故认为Survivin是潜在的治疗原发性肝癌侵袭、转移的靶标。
4.原发性肝癌中Survivin高表达可以上调H-ras基因表达水平,而H-ras基因可以通过调控OPN启动子活性,上调OPN基因表达,Survivin、H-ras和OPN三者可能存在密切的联系,并且发现Survivin促进OPN启动子活性的作用可以被H-ras基因下调所阻断。故认为,Survivin促进肝癌侵袭转移作用的部分机制可能是通过促进H-ras基因表达,激活OPN基因转录实现的。
创新点
1.发现并证实Survivin参与肝癌侵袭转移过程,为预测和治疗原发性肝癌转移复发提供了新靶标。
2.初步探索靶向Survivin的RNA干扰对肝癌细胞体外生长、侵袭、黏附力的影响及抑制体内成瘤、转移的治疗效果。
3.初步探索了Survivin促进肝癌侵袭转移的作用机制,提示Survivin、H-ras和OPN三者可能存在密切的联系,Survivin促进肝癌侵袭转移作用的部分机制可能与Survivin促进H-ras基因表达,激活OPN基因转录有关。
潜在应用价值
1.Survivin是抑制肝癌复发转移的潜在治疗靶点。
2.为深入研究Survivin参与肝癌侵袭转移的机制提供实验基础。
Hepatocellular carcinoma(HCC) is the second most common cause of death from cancer in China,which accounts for 18.8 percents of the number of total death of patients with malignant tumors.A large population with hepatitis virus B or cirrhosis attribute to the higher incidence.Surgical resection remains an important approach to a curable outcome of hepatocellular carcinoma,however,recurrence and metastasis are quite common after curative resection,which are main obstacles to successful treatment. Elucidating molecular mechanisms of HCC recurrence/metastasis and searching for effective target therapies are the promising pathway to improve the survival.
Survivin was discovered by hybridization screening of a human genomic library with the cDNA of the effector cell protease receptor-1(EPR-1).The survivin gene spans 14,700bp,and is located on chromosome 17 at band q25.The coding strand ofsurvivin encodes a protein of 142 amino acids,with a molecular weight of approximately 16.5 kDa.Survivin is a protein that inhibits apoptosis and regulates cell division.At present, more and more researches have shown that survivin can be functionally expressed in tumor cells,particularly in those with high metastasis potentials.Some research has found that stronger expression of survivin in patients with metastasis than in those without metastasis.The expression of survivin in HCC had no relationship with the patients' age,gender and differentiation level of HCC,while it was related to the intrahepatic metastasis and portal vein tumor thrombi of HCC.However,the research on the relationship of survivin and the hepatocellular carcinoma invasion and metastasis is few and the mechanisms of survivin promoting HCC recurrence/metastasis are poorly understood.
Here,we compared systematically survivin expression in HCC cell lines with differential metastasis potential and HCC specimens with or without intrahepatic metastasis,studied the relationship between survivin and HCC invasion and metastasis. By constructed and transfected the pEGFP-Cl/survivin plasmid,we studied the effects of over-expression of survivin gene on the phenotypes of human hepatocellular carcinoma cells.By constructed and transfected the pGPU6/GFP/Neo-shRNA plasmid, we studied the effects of survivin shRNA on HCCLM6 growth,adhesion,invasion in vitro,and tumor growth and metastasis behavior in vivo.And we studied the mechanisms of survivin promoting HCC malignant phenotypes.
Part one:Survivin expression in HCC cell lines with different metastatic potential and HCC samples
The main aim of this part was to study survivin expression in HCC cell lines with different metastatic potential and HCC samples,and to study the relationship between survivin expression and HCC invasion and metastasis.RT-PCR and Western Blot were applied to detect survivin expression in different metastatic potential HCC cell lines SMMC-7721,MHCC97-L,MHCC97-H,HCCLM3 and HCCLM6.Thirty-five HCC specimens with their neighboring noncancerous tissues were obtained(15 tissues with intrahepatic metastasis and 20 tissues without intrahepatic metastasis).RT-PCR and Western Blot were used to test survivin expression in tissues.Results of RT-PCR showed survivin mRNA expression gradually increased with increasing metastatic potential of cell lines.The expression of survivin mRNA was the highest in the cell line HCCLM6.Survivin mRNA expression was detected in HCC tissues.In contrast,no expression of survivin was detected in corresponding noncancerous tissues.The relative amount of survivin mRNA expression in cases of intrahepatic metastasis was 1.082±0.213,higher than that without intrahepatic metastasis(0.799±0.224,P＜0.05). Western Blot results were coincident with RT-PCR results.The levels of survivin protein in cases of intrahepatic metastasis(0.892±0.198) were significantly higher than those without intrahepatic metastasis(0.342±0.237,P＜0.05).The above experiments showed that the expression of survivin in HCC might have close relationship with invasion and metastasis.The high expression of survivin might act as an important index to indicate the process of the invasion and metastasis in HCC.
Part two:Effects of over-expression of survivin gene on the malignant phenotypes of human hepatocellular carcinoma cells
The main aim of this part was to study the effects of over-expression of survivin gene on the phenotypes of human hepatocellular carcinoma cells.Firstly,we constructed the pEGFP-C1/survivin plasmid.SMMC-7721 cells with low invasion and no metastatic potential were transfected by recombinant with empty plasmid as the control group. SMMC-7721 cells stably expressing high level survivin were screened by G418 while cells transfected with empty plasmid were obtained as the control group.Survivin expression was testified by Realtime PCR and Western Blot.Functional assays in vitro were performed to observe changes of malignant phenotypes after transfection. Eighteen nude mice with orthotropic implantation of SMMC-7721 cells were randomly equally assigned to three groups as the following:pEGFP-C1/survivin group,empty plasmid control group and PBS group.Drug administration was started at the second day after tumor implantation.50μg plasmid or PBS was injected through intraperitoneum(i.p.) once every three days for 12 times.After six weeks,mice were sacrificed to observe the tumor growth and metastasis.Survivin and OPN expression was assessed by RT-PCR and Western Blot.Pulmonary metastases were detected by H.E.stains.Differences of metastasis-related gene expression between C1 and C2 were compared by gene chips.Realtime PCR and ELISA were used to testify the different expression of H-ras and OPN.The results showed that survivin expression of SMMC-7721 cells was elevated after transfected pEGFP-C1/survivin.Functional assays in vitro indicated that C1 cells showed higher adhesive,migrant and invasive capabilities(cell adhesion rate:65.33%±7.24%vs 45.34%±5.52%;cells of outer surface in migrant assay:15.3±2.34 vs 5.3±2.53;cells in invasive assay:7.3±1.34 vs 4.3±1.25,P＜0.05),and the ability of C1 cells proliferation were higher than C2 cells. Nude mice experiments showed that tumor weight of pEGFP-C1/survivin group (2.72g±0.16g) was more than the empty plasmid control group(1.79g±0.08g,P＜0.05) and PBS group(1.75g±0.07g,P＜0.05).Similarly,the tumor volume of pEGFP-C1/survivin group was more than the empty plasmid control group and PBS group.Five mice in pEGFP-C1/survivin group were observed pulmonary metastasis. Three mice in pEGFP-C1/survivin group were observed intrahepatic metastasis.There are no mice with pulmonary/intrahepatic metastasis in control group and PBS group. The expression levels of survivin and OPN were elevated in pEGFP-C1/survivin group. The results of gene chip showed that 16 metastasis-related genes were up-regulated in SMMC-7721 cells with high survivin expression(H-ras,CTNNA1,CTNNB1,IL8RB, MMP3,ITGB3,etc),while 2 genes were down-regulated(TCF20 and TP53).Realtime PCR and ELISA found the expression of H-ras or OPN in C 1 cells was higher than C2 cells.The above experiments showed that survivin might promote the malignant phenotypes of SMMC-7721 cells.The high expression of survivin might change the expression levels of some tumor metastasis-related genes.
Part three:Study on survivin RNA interference in vitro and in vivo
The main aim of this part was to verify the effects ofsurvivin gene on the phenotypes of human hepatocellular carcinoma cells.And evaluate the effects of survivin RNAi on HCCLM6 cells growth,adhesion,invasive abilities in vitro,and tumor growth and metastasis behavior in vivo.Firstly,we designed and screened the effective survivin small interference RNA to down-regulate survivin gene expression in HCCLM6 cells. Realtime PCR and Western Blot were used to verify the RNA interference effects. According to the screening results,we constructed the pGPU6/GFP/Neo-shRNA plasmid.The recombinants were transiently transfected into HCCLM6 cells while cells transfected with control plasmid were control group.Realtime PCR and Western Blot were used to verify the RNA interference effects.The shRNA with the higher interference effect was used in the experiments in vitro and vivo.Functional assays in vitro were performed to observe changes of malignant phenotypes after transfection. Realtime PCR and ELISA were used to testify the different expression of H-ras and OPN after transfected shRNA1 plasmid.Nude mice were used to observe the effects of survivin RNAi on growth and metastasis of liver cancer in vivo.Realtime PCR and Western Blot were used to detect survivin and OPN expression.Results showed that all designed siRNA of survivin gene had some degree interference.Realtime PCR and Western Blot indicated that siRNA-166 and siRNA-358 had stronger interference effects.So we constructed respectively the shRNA1 and shRNA2 plasmid and transfected into HCCLM6 cells.Realtime PCR results showed interference efficiencies of shRNA1 and shRNA2 were respectively 78%and 65%.Western Blot results showed interference efficiencies of shRNA1 and shRNA2 were respectively 75%and 68%.So, shRNA1 was used in the followed experiments.HCCLM6 cells transfected with shRNA1 for 48h showed significantly decrease in the migrant,invasive,adhesive abilities(cell adhesion rate:43.28%±0.85%vs 55.09%±1.45%;cells of outer surface in migrant assay:15.8±0.96 vs 23.5±1.29;cells in invasive assay:10.5±1.29 vs 20.5±1.29,P＜0.01).Realtime PCR and ELISA found the levels of H-ras and OPN expression were lower than the control group when survivin expression down-regulated (P＜0.01).Nude mice experiments showed that tumor weight and volume of shRNA1 group(1.43g±0.20g/679.22mm~3+328.72mm~3) was less than the control group (2.19g±0.37g/1397.03mm~3+374.49mm~3,P＜0.01) and PBS group(2.34g±0.55g/ 1636.50 mm~3±508.80mm~3,P＜0.01).The incidence of lung metastasis of nude mice treated by shRNAI was significantly decreased(2/6) compared with the groups treated by control plasmid and PBS(6/6).The life span of nude mice treated by shRNA1 was significantly longer than the other two groups.The expression levels of survivin and OPN were lower in shRNA1 group than those in the control group.The above experiments suggested that survivin may take part in the growth and metastasis of primary liver cancer by promoting the proliferation of cancer cells,increasing the adhesive and invasive abilities.In addition,the down-regulation of survivin gene could decrease cells invasive and metastatic potential in vitro,induce the tumor growth inhibition and decrease metastatic potential in vivo.So,survivin might be a potential molecular target for invasion and metastasis of HCC therapy.
Part four:Mechanisms of survivin promoting the malignant phenotypes of human hepatocellular carcinoma
The results of the second and third part had showed that OPN and H-ras gene up-regulated when survivin was up-regulated,OPN and H-ras gene down-regulated when survivin was down-regulated.The main aim of this part was to study the relationship among survivin,H-ras and OPN,and study the mechanisms of survivin promoting HCC invasion and metastasis.Firstly,we constructed respectively pcDNA3.1(-)/survivin and pGPU6/shRNA recombinant plasmids.The plasmids were respectively transfected into HCC cells and the changes of survivin expression were measured by Realtime PCR.We subcloned the OPN gene promoter sequence into the luciferase reporter gene vector(pGL3-Basic) to construct the recombinant plasmid and designated pOPNGL3.Designed and screened the effective H-ras siRNA,and used the RNA interference to down-regulate H-ras gene expression in HCCLM6 cells.With lipofectamine mediated co-transfecion technique,pcDNA3.1(-)/survivin and pOPNGL3 were cotransfected into SMMC-7721 cells.The relative activities(Luc/β-gal) were subsequently measured to evaluate the effects of survivin over-expression on the activity of OPN gene promoter,pGPU6/shRNA and pOPNGL3 were cotransfected into HCCLM6 cells and evaluated the effects of survivin shRNA on the activity of OPN gene promoter.H-ras siRNA and pOPNGL3 were cotransfected into HCCLM6 cells and evaluated the effects of H-ras on the activity of OPN gene promoter. pcDNA3.1(-)/survivin,H-ras siRNA and pOPNGL3 were cotransfected into HCCLM6 cells,pcDNA3.1(-)/survivin,siRNA-NC and pOPNGL3 were cotransfected as the first control group,and pcDNA3.1(-),siRNA-NC and pOPNGL3 were cotransfected into HCCLM6 cells as the second control group.The relative activities of luciferase were measured to evaluate the diference of OPN gene promoter in three groups.The sequencing results confirmed that pcDNA3.1(-)/survivin and pGPU6/shRNA were correct.Survivin mRNA expression of SMMC-7721 cells was elevated after transiently transfected with pcDNA3.1(-)/survivin.Survivin mRNA expression of HCCLM6 cells was down-regulated by 70.5%after transiently transfected with pGPU6/survivin.The sequencing result confirmed that the recombinant plasmid pOPNGL3 was constructed correctly.Four designed H-ras siRNA had some degree interference after transfected HCCLM6 cells.Realtime PCR verified v-H-ras-282 has strongest interference effects (75.3%).Luciferase activity assay results showed OPN gene promoter activity increased by 187.76%after survivin over-expression in SMMC-7721 cells.OPN gene promoter activity was decreased by 61.3%after down-regulating survivin in HCCLM6 cells.OPN gene promoter activity was decreased by 59.5%after transfected H-ras siRNA.RLU of HCCLM6 cells cotransfected with pcDNA3.1(-)/survivin,H-ras siRNA and pOPNGL3 was similar with RLU of cells cotransfected with pcDNA3.1(-) and siRNA-NC(P＞0.05),was lower than cells cotransfected with pcDNA3.1(-)/survivin and siRNA-NC(P＜0.01).The above experiments suggested that survivin over-expression could promote OPN gene promoter activity.H-ras gene could regulate OPN gene promoter activity to up-regulate OPN gene expression.The effect of survivin promoting OPN gene promoter activity could be intercepted by H-ras RNAi.The relationship among survivin,H-ras and OPN might be close.Survivin might promote H-ras gene up-regulation to activate OPN gene transcription.It might be one of the mechanisms of survivin promoting HCC invasion and metastasis.
Conclusions
1.Survivin expression was associated with invasive and metastatic abilities of HCC cell lines,and with HCC intrahepatic metastasis.
2.Survivin might promote the malignant phenotypes of SMMC-7721 cells.The high expression of survivin might change the expression levels of some tumor metastasis-related genes.
3.Down-regulating survivin gene expression could decrease invasive and metastatic potential in vitro,and induce the tumor growth inhibition and decreased metastatic potential in vivo.So,survivin might be a potential molecular target for invasion and metastasis of HCC therapy.
4.The experiments showed that survivin over-expression could up-regulate H-ras gene expression level.H-ras gene could regulate OPN promoter activity to up-regulate OPN gene expression,and the effect of survivin promoting OPN gene promoter activity could be intercepted by H-ras RNAi.So,survivin might promote H-ras gene up-regulate to activate OPN gene transcription.It might be one of the mechanisms of survivin promoting HCC invasion and metastasis.
Novelty
1.Find and verify that survivin might take part in the metastasis of primary liver cancer.Survivin might be a potential molecular target for invasion and metastasis of HCC.
2.Evaluate the effects of survivin RNAi on hepatocellular carcinoma cells growth, adhesion,invasive activities in vitro,and tumor growth and metastasis behavior in vivo.
3.Preliminarily explore the mechanisms of survivin promoting HCC invasion and metastasis.The results showed that the relationship among survivin,H-ras and OPN might be close.Survivin might promote H-ras gene up-regulate to activate OPN gene transcription.It might be one of the mechanisms of survivin promoting HCC invasion and metastasis.
Potential merits for clinical application
1.Survivin may be the new therapeutic target of anti-recurrence/metastasis for HCC.
2.To supply the experimental basis for deep research the mechanisms of survivin promoting HCC invasion and metastasis.
引文
[1]Tang ZY,Ye SL,Liu YK,et al.A decade's studies on metastasis of hepatocellular carcinoma[J].J Cancer Res Clin Oncol,2004,130(4):187-196.
[2]Jemal A,Siegel R,Ward E,et al.Cancer statistics,2006[J].CA Cancer J Clin,2006,56(2):106-130.
[3]张思维,李连弟,鲁凤珠,等.中国1990-1992年原发性肝癌死亡调查分析[J].中华肿瘤杂志,1999,21(4):245-249.
[4]汤钊猷.肿瘤转移复发的基础与临床[M].上海:上海科技教育出版社,2003:1-24.
[5]Tang ZY.Hepatocellular carcinoma surgery--review of the past and prospects for the 21st century[J].J Surg Oncol,2005,91(2):95-96.
[6]Tang ZY.Treatment of hepatocellular carcinoma[J].Digestion,1998,59(5):556-562.
[7]Zhou XD,Tang ZY,Yang BH,et al.Experience of 1000 patients who underwent hepatectomy for small hepatocellular carcinoma[J].Cancer,2001,91(8):1479-1486.
[8]Tang Z,Zhou X,Lin Z,et al.Surgical treatment of hepatocellular carcinoma and related basic research with special reference to recurrence and metastasis[J].Chin Med J(Engl),1999,112(10):887-891.
[9]汤钊猷.肿瘤转移复发的基础与临床[M].上海:上海科技教育出版社,2003:289-293.
[10]Adida C,Crotty PL,McGrath J,et al.Developmentally regulated expression of the novel cancer anti-apoptosis gene survivin in human and mouse differentiation[J].Am J Pathol,1998,152(1):43-49.
[11]Ambrosini G,Adida C,Sirugo G,et al.Induction of apoptosis and inhibition of cell proliferation by survivin gene targeting[J].J Biol Chem,1998,273(18):11177-11182.
[12]Conway EM,Pollefeyt S,Cornelissen J,et al.Three differentially expressed survivin cDNA variants encode proteins with distinct antiapoptotic functions[J].Blood,2000,95(4):1435-1442.
[13]LaCasse EC,Baird S,Korneluk RG,et al.The inhibitors of apoptosis(IAPs) and their emerging role in cancer[J].Oncogene,1998,17(25):3247-3259.
[14]Jiang X,Wilford C,Duensing S,et al.Participation of Survivin in mitotic and apoptosis activities of normal and tumor-derived cells[J].J Cell Biochem,2001,83(2):342-354.
[15]Zhao J,Tenev T,Martins LM,et al.The ubiquitin-proteasome pathway regulates survivin degradation in a cell cycle-dependent manner[J].J Cell Sci,2000,113(23):4363-4371.
[16]Tamm I,Wang Y,Sausville E,et al.IAP-family protein survivin inhibits caspase activity and apoptosis induced by Fas(CD95),Bax,caspases,and anticancer drugs[J].Cancer Res,1998,58(23):5315-5320.
[17]Suzuki A,Ito T,Kawano H,et al.Survivin initiates procaspase 3/p21 complex formation as a result of interaction with Cdk4 to resist Fas-mediated cell death[J]. Oncogene, 2000, 19(10): 1346-1353.
[18] Li F, Ambrosini G, Chu EY, et al. Control of apoptosis and mitotic spindle checkpoint by survivin[J]. Nature, 1998, 396(6711): 580-584.
[19] Giodini A, Kallio MJ, Wall NR, et al. Regulation of microtubule stability and mitotic progression by survivin[J]. Cancer Res, 2002, 62(9): 2462-2467.
[20] O'Connor DS, Schechner JS, Adida C, et al. Control of apoptosis during angiogenesis by survivin expression in endothelial cells[J]. Am J Pathol, 2000, 156(2): 393-398.
[21] Lee GH, Joo YE, Koh YS, et al. Expression of survivin in gastric cancer and its relationship with tumor angiogenesis[J]. Eur J Gastroenterol Hepatol, 2006, 18(9): 957-963.
[22] Miyachi K, Fujita M, Tanaka N, et al. Correlation between telomerase activity and telomeric-repeat binding factors in gastric cancer[J]. J Exp Clin Cancer Res, 2002, 21(2): 269-275.
[23] Tsuburaya A, Noguchi Y, Yoshikawa T, et al. An anti-apoptosis gene, survivin and telomerase expression in gastric cancer[J]. Hepatogastroenterology, 2002, 49(46): 1150-1152.
[24] Zhu H, Chen XP, Zhang WG, et al. Expression and significance of new inhibitor of apoptosis protein survivin in hepatocellular carcinoma[J]. World J Gastroenterol, 2005, 11(25): 3855-3859.
[25] Ikeguchi M, Hirooka Y, Kaibara N. Quantitative analysis of apoptosis-related gene expression in hepatocellular carcinoma[J]. Cancer, 2002, 95(9): 1938-1945.
[1]Chakravarti A,Noll E,Black PM,et al.Quantitatively determined survivin expression levels are of prognostic value in human gliomas[J].J Clin Oncol,2002,20(4):1063-1068.
[2]Kleinberg L,Fl(?)renes VA,Silins I,et al.Nuclear expression of survivin is associated with improved survival in metastatic ovarian carcinoma[J].Cancer.2007,109(2):228-238.
[3]Yie SM,Luo B,Ye NY,et al.Detection of Survivin-expressing circulating cancer cells in the peripheral blood of breast cancer patients by a RT-PCR ELISA[J].Clin Exp Metastasis,2006,23(5-6):279-289.
[4]Zhu H,Chen XP,Zhang WG,et al.Expression and significance of new inhibitor of apoptosis protein survivin in hepatocellular carcinoma[J].World J Gastroenterol,2005,11(25):3855-3859.
[5]Ikeguchi M,Hirooka Y,Kaibara N.Quantitative analysis of apoptosis-related gene expression in hepatocellular carcinoma[J].Cancer,2002,95(9):1938-1945.
[6]Tian J,Tang ZY,Ye SL,et al.New human hepatocellular carcinoma(HCC) cell line with highly metastatic potential(MHCC97) and its expression of the factors associated with metastasis[J].Br J Cancer,1999,81(5):814-821.
[7]Li Y,Tang ZY,Ye SL,et al.Establishment of cell clones with different metastatic potential from the metastatic hepatocellular carcinoma cell line MHCC97[J].World J Gastroenterol,2001,7(5):630-636.
[8]Ambrosini G,Adida C,Sirugo G,et al.Induction of apoptosis and inhibition of cell proliferation by survivin gene targeting[J].J Biol Chem,1998,273(18):11177-11182.
[9]Tamm I,Wang Y,Sausville E,et al.IAP family protein survivin inhibits caspase activity and apoptosis induced by Fas(CD95),Bax,caspases,and anticancer drugs[J].Cancer Res,1998,58(23):5315-5320.
[10]Ikeguchi M,Hirooka Y,Kaibara N.Quantitative analysis of apoptosis-related gene expression in hepatocellular carcinoma[J].Cancer,2002,95(9):1938-1945.
[11]戴德坚,吴丹,孟化,等.反义survivin-脂质体复合物对肝癌细胞生长凋亡和细胞周期的影响[J].Chin J Oncol,2005,27(10):581-585.
[12]Fields AC,Cotsonis G,Sexton D,et al.Survivin expression in hepatocellular carcinoma:correlation with proliferation,prognostic parameters,and outcome.Mod Pathol[J].2004,17(11):1378-1385.
[13]Zhu H,Chen XP,Zhang WG,et al.Expression and significance of new inhibitor of apoptosis protein survivin in hepatocellular carcinoma[J].World J Gastroenterol.2005,11(25):3855-3859.
[14]Ye CP,Qiu CZ,Huang ZX,et al.Relationship between survivin expression and recurrence,and prognosis in hepatoceilular carcinoma[J].World J Gastroenterol.2007,13(46):6264-6268.
[1]Ambrosini G,Adida C,Altieri DC.A novel anti-apoptosis gene,survivin,expressed in cancer and lymphoma[J].Nat Med,1997,3(8):917-921.
[2]Adida C,Crotty PL,McGrath J,et al.Developmentally regulated expression of the novel cancer anti-apoptosis gene survivin in human and mouse differentiation[J].A m J Pathol,1998,152(1):43-49.
[3]Ito T,Shiraki K,Sugimoto K,et al.Survivin promotes cell proliferation in human hepatocellular carcinoma[J].Hepatology,2000,31(5):1080-1085.
[4]Sarela AI,Macadam RC,Farmery SM,et al.Expression of the antiapoptosis gene,survivin,predicts death from recurrent colorectal carcinoma[J].Gut,2000,46(5):645-650.
[5]Okada E,Murai Y,Matsui K,et al.Survivin expression in tumor cell nuclei is predictive of a favorable prognosis in gastric cancer patients[J].Cancer Lett,2001,163(1):109-116.
[6]Lu CD,Altieri DC,Tanigawa N.Expression of a novel antiapoptosis gene,survivin,correlated with tumor cell apoptosis and p53 accumulation in gastric carcinomas[J].Cancer Res,1998,58(9):1808-1812.
[7]Adida C,Berrebi D,Peuchmaur M,et al.Anti-apoptosis gene,survivin,and prognosis of neuroblastoma[J]. Lancet, 1998, 351(9106): 882-883.
[8] Livak KJ, Schmittgen TD. Analysis of relative gene expression data using realt-time quantitative PCR and the 2(-Delta Delta C(T)) Method[J]. Methods, 2001, 25(4): 402-408.
[9] Sarela AI, Macadam RC, Farmery SM, et al. Expression of the antiapoptosis gene, survivin, predicts death from recurrent colorectal carcinoma[J]. Gut 2000, 46(5): 645-650.
[10] Mori A, Wada H, Nishimura Y, et al. Expression of the antiapoptosis gene survivin in human leukemia[J]. Int J Hematol, 2002, 75(2): 161-165.
[11] Das A, Tan WL, Teo J, et al. Expression of survivin in primary glioblastomas[J]. J Cancer Res Clin Oncol, 2002, 128(6): 302-306. Epub 2002 Apr 24.
[12] Ito T, Shiraki K, Sugimoto K, et al. Survivin promotes cell proliferation in human hepatocellular carcinoma[J]. Hepatology, 2000, 31(5): 1080-1085.
[13] Sarela AI, Verbeke CS, Ramsdale J, et al. Expression of survivin, a novel inhibitor of apoptosis and cell cycle regulatory protein, in pancreatic adenocarcinoma[J]. Br J Cancer, 2002, 86(6): 886-892.
[14] Wakana Y, Kasuya K, Katayanagi S, et al. Effect of survivin on cell proliferation and apoptosis in gastric cancer[J]. Oncol Rep, 2002, 9(6): 1213-1218.
[15] Cohen C, Lohmann CM, Cotsonis G, et al. Survivin expression in ovarian carcinoma: correlation with apoptotic markers and prognosis[J]. Mod Pathol 2003, 16(6): 574-583.
[16] Lu CD, Altieri DC, Tanigawa N. Expression of a novel antiapoptosis gene, survivin, correlated with tumor cell apoptosis and p53 accumulation in gastric carcinomas[J]. Cancer Res, 1998, 58(9): 1808-1812.
[17] Trieb K, Lehner R, Stulnig T, et al. Survivin expression in human osteosarcoma is a marker for survival[J]. Eur J Surg Oncol, 2003, 29(4): 379-382.
[18] Scharovsky OG, Rozados VR, Gervasoni SI, et al. Inhibition of ras oncogene: a novel approach to antineoplastic therapy[J]. J Biomed Sci, 2000, 7(4): 292-298.
[19] Boivin-Angele S, Lefrancois L, Froment O, et al. Ras gene mutations in vinyl chloride-induced liver tumours are carcinogen-specific but vary with cell type and species[J]. Int J Cancer, 2000, 85(2): 223-227.
[20] Tsunematsu S, Saito H, Sato R, et al. Role of H-ras gene in chronic liver damage in mice. By using transgenic mice carrying a human C-H-ras proto-oncogene without mutations[J]. Biochem Mol Biol Int, 1997. 42(2): 371-379.
[21] Cochet O, Kenigsberg M, Delumeau I. et al. Intracellular expression of an antibody fragment-neutralizing p21 ras promotes tumor regression[J]. Cancer Res, 1998,58(6): 1170-1176.
[22] Fujita M, Norris DA, Yagi H, et al. Overexpression of mutant ras in human melanoma increases invasiveness, proliferation and anchorage-independent growth in vitro and induces tumour formation and cachexia in vivo[J]. Melanoma Res, 1999, 9(3): 279-291.
[23] Kim MS, Son MW, Kim WB, et al. Apicidin, an inhibitor of histone deacetylase, prevents H-ras induced invasive phenotype[J]. Cancer Lett, 2000, 157(1): 23-30.
[24] Weijzen S, Velders MP, Kast WM. Modulation of the immune response and tumor growth by activated Ras[J]. Leukemia, 1999, 13(4): 502-513.
[25] Chin L, Tarn A, Pomerantz J, et al. Essential role for oncogenic Ras in tumour maintenance[J]. Nature, 1999, 400(6743): 468-472.
[26] Guo HB, Zhang QS, Chen HL. Effects of H-ras and v-sis overexpression on N-acetylglucosaminyltransferase V and metastasis-related phenotypes in human hepatocarcinoma cells[J]. J Cancer Res Clin Oncol, 2000, 126(5): 263-270.
[27] Wang Q, Lin ZY, Feng XL. Alterations in metastatic properties of hepatocellular carcinoma cell following H-ras oncogene transfection[J]. World J Gastroenterol, 2001, 7(3): 335-339.
[28] 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[J]. Cancer, 2003, 98(1): 119-127.
[29] Ye QH, Qin LX, Forgues M, et al. Predicting hepatitis B virus-positive metastatic hepatocellular carcinomas using gene expression profiling and supervised machine learning[J]. Nat Med, 2003, 9(4): 416-423. Epub 2003 Mar 17.
[1]Tang ZY.Hepatocellular carcinoma surgery--review of the past and prospects for the 21st century[J].J Surg Oncol,2005,91(2):95-96.
[2]Caplen NJ.A new approach to the inhibition of gene expression[J].Trends Biotechnol,2002,20(2):49-51.
[3]Montgomery MK,Xu S,Fire A.RNA as a target of double-stranded RNA-mediated genetic interference in Caenorhabditis elegans[J].Proc Natl Acad Sci U S A,1998,95(26):15502-15507.
[4]王伟,朱焕章,薛京伦.RNA干扰在基因治疗中的应用[J].生物化学与生物物理进展,2004,31(7):590-594.
[5]Biroccio A,Leonetti C,Zupi G.The future of antisense therapy:combination with anticancer treatments[J].Oncogene,2003,22(42):6579-6588.
[6]Tian J,Tang ZY,Ye SL,et al.New human hepatocellular carcinoma(HCC) cell line with highly metastatic potential(MHCC97) and its expression of the factors associated with metastasis[J].Br J Cancer,1999,81(5):814-821.
[7]闫歌,黄爱龙,唐霓,等.短发夹状RNA抑制survivin基因在肝癌细胞中的表达[J].中华肝脏病杂志,2003,11(12):712-715.
[8]Pennati M,Colella G,Folini M,et al.Ribozyme-mediated attenuation of survivin expression sensitizes human melanoma cells to cisplati-ninduced apoptosis[J].J Clin Invest,2002,109(2):285-286.
[9]Cao C,Mu Y,Hallahan DE,et al.XIAP and survivin as therapeutic targets for radiation sensitization in preclinical models of lung cancer[J].Oncogene,2004,23(42):7047-7052.
[10]McKay TR,Bell S,Tenev T,et al.Procaspase 3 expression in ovarian carcinoma cells increases survivin transcription which can be countered with a dominant-negative mutant,survivin T34A;a combination gene therapy strategy[J].Oncogene,2003,22(23):3539-3547.
[11]Carthew RW.Gene silencing by double-stranded RNA[J].Curr Opin Cell Biol,2001,13(2):244-248.
[12]Pushparaj PN,Melendez AJ.Short interfering RNA(siRNA) as a novel therapeutic[J].Clin Exp Pharmacol Physiol,2006,33(5-6):504-510.
[13]Aigner A.Gene silencing through RNA interference(RNAi) in vivo:strategies based on the direct application of siRNAs[J].J Biotechnol,2006,124(1):12-25.
[14]Aoki Y,Cioca DP,Oidaira H,et al.RNA interference may be more potent than antisense RNA in human cancer cell lines[J].Clin Exp Pharmacol Physiol.2003,30(1-2):96-102.
[15]Lee NS,Dohjima T,Bauer G,et al.Expression of small interfering RNAs targeted against HIV-1 rev transcripts in human cells[J].Nat Biotechnol,2002,20(5):500-505.
[16]Miyagishi M,Taira K.U6 promoter-driven siRNAs with four uridine 3'overhangs efficiently suppress targeted gene expression in mammalian cells[J].Nat Biotechnol.2002,20(5):497-500.
[17]Luo KQ,Chang DC.The gene-silencing effciency of siRNA is strongly dependent on the local structure of mRNA at the targeted region[J].Biochem Biophys Res Commun,2004,318(1):303-310.
[18]Overhoff M,Alken M,Far RK,et al.Local RNA target structure influences siRNA efficacy:a systematic global analysis[J].J Mol Biol,2005,348(4):871-881.
[19]Kappler M,Bache M,Barrel F,et al.Knockdown of survivin expression by small interfering RNA reduces the clonogenic survival of human sarcoma cell lines independently of P53[J].Cancer Gene Ther,2004,11(3):186-193.
[20]卢昕,郑启昌,熊俊,等.siRNA抑制肝癌细胞株HepG2 survivin基因表达的研究[J].华中科技大学学报(医学版),2004,33(6):696-699.
[1]Gotoh M,Sakamoto M,Kanetaka K,et al.Overexpression of osteopontin in hepatocellular carcinoma[J].Pathol Int,2002,52(1):19-24.
[2]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[J].Cancer,2003,98(1):119-127.
[3]Ye QH,Qin LX,Forgues M,et al.Predicting hepatitis B virus-positive metastatic hepatocellular carcinomas using gene expression profiling and supervised machine learning[J].Nat Med,2003,9(4):416-423.
[4]Guo HB,Zhang QS,Chen HL.Effects of H-ras and v-sis overexpression on N-acetylglucosaminyltransferase V and metastasis-related phenotypes in human hepatocarcinoma cells[J].J Cancer Res Clin Oncol,2000,126(5):263-270.
[5]Teramoto H,Castellone MD,Malek RL,et al.Autocrine activation of an osteopontin-CD44-Rac pathway enhances invasion and transformation by H-RasV 12[J].Oncogene,2005,24(3):489-501.
[6]Denhardt DT,Mistretta D,Chambers AF,et al.Transcriptional regulation of osteopontin and the metastatic phenotype:evidence for a Ras-activated enhancer in the human OPN promoter[J].Clin Exp Metastasis,2003,20(1):77-84.
[7]Zuber J,Tchernitsa OI,Hinzmann B,et al.A genome-wide survey of RAS transformation targets[J].Net Genet,2000,24(2):144-152.
[8]Weijzen S,Velders MP,Kast WM.Modulation of the immune response and tumor growth by activated ras[J].Leukemia,1999,13(4):502-513.
[9]汪青,林芷英,冯晓黎.H-ras癌基因转染后SMMC-7721细胞转移特性的体内外检测[J].中国临床医学,2001,8(2):133-134.
[10]Weber GF,Ashkar S,Glimcher MJ,et al.Receptor-ligand interaction between CD44 and osteopontin(Eta-1)[J].Science,1996,271(5248):509-512.
[11]Denhardt DT,Giachelli CM,Rittling SR.Role of osteopontin in cellular signaling and toxicant injury[J].Annu Rev Pharmacol Toxicol,2001,41:723-749.
[12]Oates AJ,Barraclough R,Rudland PS,et al.The identification of osteopontin as a metastasis-related gene product in a rodent mammary tumour model[J].Oncogene,1996,13(1):97-104.
[13]Urquidi V,Sloan D,Kawai K,et al.Contrasting expression of thrombospondin-1and osteopontin correlates with absence or presence of metastatic phenotype in an isogenic model of spontaneous human breast cancer metastasis[J].Clin Cancer Res, 2002, 8(1): 61-74.
[14] Frederick M, Ausubel, Roger Brent, et al. Current protocols in molecular biology[M]. 1998; John Wiley&Sons, Inc.. p9.6.1, p9.6.6.
[15] Chambers AF, Behrend EI, Wilson SM, et al. Induction of expression of osteopontin (OPN; secreted phosphoprotein) in metastatic, ras-transformed NIH3T3 cells[J]. Anticancer Res, 1992, 12(1): 43-47.
[1]Ambosini G,Adida G,Altieri DC.A novel anti-apotosis gene,survivin,expressed in cancer and lymphoma[J].Nat Med,1997,3(8):917-921.
[2]Adida C,Crotty PL,McGrath J,et al.Developmentally regulated expression of the nover cancer anti-apoptosis gene survivin in human and mouse differentiation [J].Am J Pathol,1998,152(1):43-49.
[3]Konno R,Yamakawa H,Utsunomiya H,et al.Expression ofsurvivin and Bcl-2 in the normal human endometrium[J].Mol Hum Reprod,2000,6(6):529-534.
[4]Uren AG,Beilharz T,O'connell MJ,et al.Role for yeast inhibitor of apoptosis(IAP)-like proteins in cell division[J].Proc Natl Acad Sci U S A,1999,96(18):10170-10175.
[5]Fraser AG,James C,Evan GI,et al.Caenorhabditis elegans inhibitor of apotosis protein(IAP) homologue BIR-1 plays a conserved role in cytokinesis[J].Curr Biol,1999,9(6):292-301.
[6]Takahashi R,Deveraux Q,Tamm I,et al.A single BIR domain of XIAP sufficient for inhibiting caspases[J].J Biol Chem,1998;273(14):7787-7790.
[7]Ambrosini G,Adida C,Sirugo G,et al.Induction of apoptosis and inhibition of cell proliferation by survivin gene targeting[J].J Biol Chem,1998,273(18):11177-11182.
[8]Li F,Altieri DC.The cancer antiapoptosis mouse survivin gene:characterization of locus and transcriptional requirements of basal and cell cycle-dependent expression[J].Cancer Res,1999,59(13):3143-3151.
[9]Li F,Ambrosini G,Chu EY,et al.Control of apoptosis and mitotic spindle checkpoint by survivin[J].Nature,1998,396(6711):580-584.
[10]Deveraux QL,Reed JC.IAP family proteins--suppressors of apoptosis[J].Genes Dev, 1999, 13(3): 239-252.
[11] LaCasse EC, Baird S, Korneluk RG, et al. The inhibitors of apoptosis (IAPs) and their emerging role in cancer[J]. Oncogene, 1998, 17(25): 3247-3259.
[12] Zwicker J, Lucibello FC, Wolfraim La, et al. Cell cycle regulation of the cyclin A, cdc25C and cdc2 genes is based on a common mechanism of transcriptional repression[J]. EMBO J, 1995, 14(18): 4514-4522.
[13] Jiang X, Wilford C, Duensing R, et al. Participation of survivin in mitotic and apoptosis activities of normal and tumor-derived cells[J]. J Cell Biochem, 2001, 83(2): 342-354.
[14] Zhao J, Tenev T, Martins LM, et al. The ubiquitin-proteasome pathway regulates survivin degradation in a cell cycle-dependent manner[J]. J Cell Sci, 2000, 113(23): 4363-4371.
[15] Rohayem J, Diestelkoetter P, Weigle B, et al. Antibody response to the tumor-associated inhibitor of apoptosis protein survivin in cancer patients[J]. Cancer Res, 2000, 60(7): 1815-1817.
[16] Hirohashi Y, Torigoe T, Maeda A, et al. An HLA-A24-restricted cytotoxic T lymphocyte epitope of a tumor-associated protein, survivin[J]. Clin Cancer Res, 2002,8(6): 1731-1739.
[17] Sarela AI, Macadam RC, Farmery SM, et al. Expression of the antiapoptosis gene, survivin, predicts death from recurrent colorectal carcinoma[J]. Gut, 2000, 46(5): 645-650.
[18] Lehner R, Enomoto T, McGregor JA, et al. Correlation of survivin mRNA detection with histologic diagnosis in normal endometrium and endometrial carcinoma[J]. Acta Obstet Gynecol Scand, 2002, 81(2): 162-167.
[19] Koch CA, Vortmeyer AO, Diallo R, et al. Survivin: a novel neuroendocrine marker for pheochromocytoma[J]. Eur J Endocrinol, 2002, 146(3): 381-388.
[20] Tamm I, Wang Y, Sausville E, et al. IAP-family protein survivin inhibits caspase activity and apoptosis induced by Fas (CD95), Bax, caspases, and anticancer drugs[J]. Cancer Res, 1998, 58(23): 5315-5320.
[21] Velculescu VE, Madden SL, Zhang L, et al. Analysis of human transcriptomes[J]. Nat Genet, 1999, 23(4): 387-388.
[22] Reed JC. Dysregulation of apoptosis in cancer[J]. J Clin Oncol, 1999, 17(9): 2941-2953.
[23]Hattori M,Sakamoto H,Satoh K,et al.DNA demethylase is expressed in ovarian cancers and the expression correlates with demethylation of CpG sites in the promoter region of c-erbB-2 and survivin genes[J].Cancer Lett,2001,169(2):155-164.
[24]Mirza A,McGuirk M,Hockenberry TN,et al.Human survivin is negatively regulated by wild-type p53 and participates in p53-dependent apoptotic pathway[J].Oncogene,2002,21(17):2613-2622.
[25]Kawasaki H,Altieri DC,Lu CD,et al.Inhibition of apoptosis by survivin predicts shorter survival rates in colorectal cancer[J].Cancer Res,1998,58(22):5071-5074.
[26]王孝养,陈仕新,张珍祥,等.survivin基因在非小细胞癌中的表达及与p53、c-myc、 k-ras蛋白表达的关系[J].中华结核和呼吸杂志,2001,24(6):371-374.
[27]Chen J,Wu W,Tahir SK,et al.Down-regulation of survivin by antisense oligonucleotides increases apoptosis,inhibits cytokinesis and anchorage-independent growth[J].Neoplasia,2000,2(3):235-241.
[28]Suzuki A,Ito T,Kawano H,et al.Survivin initiates procaspase 3/p21 complex formation as a result of interaction with Cdk4 to resist Fas-mediated cell death.[J].Oncogene 2000,19(10):1346-1353.
[29]O'Connor DS,Schechner JS,Adida C,et al.Control of apoptosis during angiogenesis by survivin expression in endothelial cells[J].Am J Pathol,2000,156(2):393-398.
[30]Mesri M,Morales-Ruiz M,Ackermann EJ,et al.Suppression of vascular endothelial growth factor-mediated endothelial cell protection by survivin targeting[J].Am J Pathol,2001,158(5):1757-1765.
[31]Papapetropoulos A,Fulton D,Mahboubi K,et al.Angiopoietin-1 inhibits endothelial cell apoptosis via the Akt/survivin pathway[J].J Biol Chem,2000,275(13):9102-9105.
[32]Falleni M,Pellegrini C,Marchetti A,et al.Survivin gene expression in early-stage non-small cell lung cancer[J].J Pathl,2003,200(5):620-626.
[33]张勤,陈志伟,王丽,等.非小细胞肺癌Survivin表达与其预后关系的研究[J].中国综合临床,2002,18(10):925-926.
[34] Grabowski P, Kuhnel T, Muhr-Wilkenshoff F, et al. Prognostic value of nuclear survivin expression in oesophageal squamous cell carcinoma[J]. Br J Cancer, 2003, 88(1): 115-119.
[35] Lu CD, Altieri DC, Tanigawa N. Expression of a novel antiapoptosis gene, Survivin, correlated with tumor cell apoptosis and p53 accumulation in gastric carcinomas[J]. Cancer Res, 1998, 58(9): 1808-1812.
[36] Lee GH, Joo YE, Koh YS, et al. Expression of survivin in gastric cancer and its relationship with tumor angiogenesis[J]. Eur J Gastroenterol Hepatol, 2006, 18(9): 957-963.
[37] Morinaga S, Nakamura Y, Ishiwa N, et al. Expression of survivin mRNA associates with apoptosis, proliferation and histologically aggressive features in hepatocellular carcinoma[J]. Oncol Rep, 2004, 12(6): 1189-1194.
[38] Ikeguchi M, Hirooka Y, Kaibara N. Quantitative analysis of apoptosis-related gene expression in hepatocellular carcinoma[J]. Cancer, 2002, 95(9): 1938-1945.
[39] Fields AC, Cotsonis G, Sexton D, et al. Survivin expression in hepatocellular carcinoma: correlation with proliferation, prognostic parameters, and outcome[J]. Mod Pathol. 2004, 17(11): 1378-1385.
[40] Zhu H, Chen XP, Zhang WG, et al. Expression and significance of new inhibitor of apoptosis protein survivin in hepatocellular carcinoma[J]. World J Gastroenterol. 2005, 11(25): 3855-3859.
[41] Ye CP, Qiu CZ, Huang ZX, et al. Relationship between survivin expression and recurrence, and prognosis in hepatocellular carcinoma[J]. World J Gastroenterol. 2007, 13(46): 6264-6268.
[42] 黎军和,何文静,何有兼．Survivin和Livin在Dukes’B期结直肠癌中的表达及其临床意义[J]．癌症,2007,26(5)：547-551．
[43] Sharp JD, Hausladen DA, Maher MG, et al. Bladder cancer detection with urinary survivin, an inhibitor of apoptosis[J]. Front Biosci, 2002, 7(1): 36-41.
[44] Ziaee SA, Moula SJ, Hosseini Moghaddam SM, et al. Diagnosis of bladder cancer by urine survivin, an inhibitor of apoptosis: a preliminary report[J]. Urol J. 2006, 3(3): 150-153.
[45] Adini A, Kornaga T, Firoozbakht F, et al. Placental growth factor is a survival factor for tumor endothelial cells and macrophages[J]. Cancer Res, 2002, 62(10): 2749-2752.
[46]Sandler A,Scott D,Azuhata T,et al.The survivin:Fas ratio is predictive of recurrent disease in neuroblastoma[J].J Pediatr Surg,2002,37(3):507-511.
[47]Ansell SM,Arendt BK,Grote DM.Inhibitor of survivin expression suppresses the growth of aggressive non-Hodgkin's lymphoma[J].Leukemia,2004,18(3):616-623.
[48]戴德坚,吴丹,孟化,等.反义survivin-脂质体复合物对肝癌细胞生长凋亡和细胞周期的影响[J].中华肿瘤杂志,2005,27(10):581-585.
[49]Kappler M,Bache M,Bartel F,et al.Knockdown of survivin expression by small interfering RNA reduces the clonogenic survival of human sarcoma cell lines independently of p53[J].Cancer Gene Ther,2004,11(3):186-193.
[50]Ling X,Li F.Silencing of antiapoptotic survivin gene by multiple approaches of RNA interference technology[J].Biotechniques,2004,36(3):450-454,456-460.
[51]Pennati M,Binda M,De Cesare M,et al.Ribozyme-mediated down-regulation of survivin expression sensitizes human melanoma cells to topotecan in vitro and in vivo[J].Carcinogenesis,2004,25(7):1129-1136.
[52]Tsuruma T,Hata F,Torigoe T,et al.Phase I clinical study ofanti-apoptosis protein,survivin-derived peptide vaccine therapy for patients with advanced or recurrent colorectal cancer[J].J Transl Med,2004,2(1):19.
[53]Xiang R,Mizutani N,Luo Y,et al.A DNA vaccine targeting survivin combines apoptosis with suppression of angiogenesis in lung tumor eradication[J].Cancer Res,2005,65(2):553-561.
[54]Fulda S,Debatin KM.Sensitization for tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis by the chemopreventive agent resveratrol[J].Cancer Res,2004,64(1):337-346.
[55]Lu B,Mu Y,Cao C,et al.Survivin as a therapeutic target for radiation sensitization in lung cancer[J].Cancer Res,2004,64(8):2840-2845.
[56]Asanuma K,Moriai R,Yajima T,et al.Survivin as a radioresistance factor in pancreatic cancer[J].Jpn J Cancer Res,2000,91(11):1204-1209.