【作者】 刘凌晓；

【导师】 王建华； 王小林； 颜志平； JM Luk；

【作者基本信息】 复旦大学 ， 影像医学与核医学， 2008， 博士

【摘要】 第一部分靶向肝肠钙黏附蛋白miR-30-based shRNA慢病毒的构建、鉴定及大量包装目的:以Ll-cadherin基因为靶标设计miR-30-based shRNA序列,构建重组慢病毒,用以研究肝肠钙黏附蛋白在肝癌生长和转移过程中的作用,并为基因干扰治疗肝癌的动物实验提供工具。方法:参照冷泉港实验室设计的miR-30-based shRNA系列库,采用Openbiosystem公司定制的以逆转录病毒为基本骨架,确认有效干扰靶基因和对照基因的PSM2系列载体,由PSM2质粒PCR制备携带miR-30-basedshRNA序列的插入片段,再提纯浓缩测序后与慢病毒线性质粒连接,构建重组pLUNIG-miLl-cadherin工作质粒。同时重组构建干扰荧光素酶基因pLUNIG-miLuciferase工作质粒作为非靶基因干扰对照。采用四质粒包装系统,重组pLUNIG-miLl-cadherin或pLUNIG-miLuciferase为工作质粒,PCMV-VSVG、PMDL-G/P-RRE、PRSV-REV为包装质粒,用磷酸钙-DNA共沉淀法转入293T细胞中包装,收集病毒上清液并浓缩,Q-RT-PCR测定病毒拷贝数。慢病毒感染MHCC97H细胞,上清液转染293T细胞排除病毒复制性。G418筛选稳定感染的MHCC97H-miLl-cadherin和MHCC97H-miLuciferase细胞株,经实时定量PCR,细胞免疫组化及Western blotting检测证实MHCC97H-miLl-cadherin细胞中Ll-cadherin的有效下调。结果:1、用BLAST同源检索进行测序结果的鉴定。成功获得命名为pLUNIG-miLl-cadherin和pLUNIG-miLuciferase的工作质粒。2、稳定感染的MHCC97H-miLl-cadherin细胞株中Ll-cadherin mRNA的表达有一定程度降低,相对值约为MHCC97H对照组的49%。细胞免疫荧光染色显示,MHCC97H-miLl-cadherin细胞中Ll-cadherin蛋白的表达与非靶基因干扰MHCC97H-miLuciferase细胞组相比明显降低。Western blotting进一步确认,MHCC97H-miLl-cadherin细胞中Ll-cadherin蛋白(120 KDa)的表达明显降低,非靶基因干扰MHCC97H-miLuciferase细胞组与无处理对照MHCC97H细胞组Ll-cadherin表达接近。3、大量包装后慢病毒浓缩原液通过实时定量PCR检测得到每批病毒拷贝的数量级均在10~9以上,且病毒无复制性。结论:1、构建了新一代双选择系统的重组慢病毒携带miR-30-based shRNA系列工作质粒,包括靶基因和荧光素酶基因干扰对照两种。证实慢病毒是携带shRNA进入肝癌细胞的理想载体。2、在基因和蛋白表达水平证实应用miR-30-based shRNA能有效地抑制高转移潜能人肝癌细胞株MHCC97H内Ll-cadherin的表达。3、完成了用于体外和活体实验的高滴度重组慢病毒的大批量包装生产、浓缩和复制性的检测。第二部分基因干扰肝肠钙黏附蛋白的人肝癌细胞株的细胞生物学属性改变目的:研究慢病毒介导的肝肠钙黏附蛋白miR-30-based shRNA稳定转染的肝癌细胞株MHCC97H-miLl-cadherin的细胞增殖、粘附、克隆形成、迁移、侵袭、细胞周期、细胞凋亡以及针对性的药物敏感性的变化。探讨其相关关键信号途径。方法:以荧光素酶干扰MHCC97H-miLuciferase细胞和无处理MHCC97H细胞作为对照组,运用吸光度测量法进行细胞增殖实验和细胞粘附实验;Pl/Rnase染色-流式细胞分析法检测细胞周期;TUNEL法检测细胞凋亡;软琼脂法进行细胞克隆形成实验;TRANSWELL法检测细胞迁移和侵袭能力;MTT法进行多种化疗药及p53基因药物敏感性实验及Western blotting进行相关信号通路的初步探讨。结果:1、靶基因干扰组MHCC97H-miLl-cadherin细胞的生长速度和粘附能力较对照组细胞明显抑制,靶基因干扰组与对照组相比差异具有显著统计学意义(P＜0.05)。2、靶基因干扰组MHCC97H-miLl-cadherin细胞与荧光素酶干扰组MHCC97H-miLuciferase细胞和无处理对照组MHCC97H细胞相比S期细胞百分比明显减少,G0-G1期细胞百分比明显增多,靶基因干扰组与对照组相比差异具有显著统计学意义(P＜0.01)。3、靶基因干扰组MHCC97H-miLl-cadherin凋亡阳性细胞为8.52±1.43%,显著高于荧光素酶干扰组MHCC97H-miLuciferase(1.55±0.33%),靶基因干扰组与对照组相比差异具有显著统计学意义(P＜0.001)。4、MHCC97H-miLl-cadherin组的细胞软琼脂克隆数明显少于MHCC97H和MHCC97H-miLuciferase组,抑制率为91.3%,靶基因干扰组与对照组相比差异具有显著统计学意义(P＜0.001)。5、迁移和侵袭实验中MHCC97H-miLl-cadherin组的穿膜细胞明显少于MHCC97H和MHCC97H-miLuciferase组,抑制率分别为90.2和88.3%,靶基因干扰组与对照组相比差异具有显著统计学意义(P＜0.001)。6、靶基因干扰组MHCC97H-miLl-cadherin细胞与荧光素酶干扰组MHCC97H-miLuciferase细胞和无处理对照组MHCC97H细胞相比针对性的化疗药物如表阿霉素、卡铂以及基因药物rAd-p53的敏感性明显增高。7、通过Wnt/β-catenin信号通路改变,Ll-cadherin的下调激活了GSK-3β的活性,从而导致β-catenin的降解,抑制了Cyclin D1的表达而相应增加Rb的表达,从而改变了MHCC97H细胞的肿瘤生物学属性。结论:1、MHCC97H-mi Ll-cadherin细胞株与对照组相比生物学属性改变明显,表现为增殖迟缓,粘附能力明显下降,软琼脂克隆形成,迁移、侵袭的能力明显减退,S期细胞百分比明显减少,G0-G1期细胞百分比明显增多,细胞自发凋亡显著增多。2、MHCC97H-mi Ll-cadherin细胞株与对照组相比对一些针对性的化疗药物如表阿霉素、卡铂的敏感性和抑制率明显增高。3、MHCC97H-mi Ll-cadherin细胞株与对照组相比对基因药物rAd-p53的敏感性和抑制率明显增高。4、人源性高转移潜能MHCC97H肝癌细胞株中Ll-cadherin的下调通过对细胞增殖和凋亡的调控明显改变了这一细胞株的肿瘤生物学属性,高度提示Ll-cadherin的癌基因特性。第三部分基因干扰肝肠钙黏附蛋白联合rAd-p53分子靶向治疗对肝癌生长、转移的抑制作用及相关信号通路的初步探讨目的:探讨靶基因干扰处理的MHCC97H-mi-Ll-cadherin细胞皮下成瘤能力、继发性转移能力以及尾静脉注射后继发性转移能力变化。研究皮下移植肝癌荷瘤鼠联合分子靶向治疗后肿瘤生长的变化、肿瘤组织中Ll-cadherin、p53及其相关信号通路的基因表达的变化。方法:将裸鼠分三组分别于皮下种植MHCC97H-mi-Ll-cadherin、非特异性基因干扰对照MHCC97H-mi-Luciferase以及无处理对照MHCC97H细胞,观察靶基因干扰处理组与对照组相比裸鼠皮下成瘤能力和皮下肿瘤转移能力的变化;同样分组尾静脉注射三组肿瘤细胞观察靶基因干扰处理组与对照组相比继发性转移能力的变化;分组重组慢病毒瘤内注射和联合P53分子靶向治疗后观察对皮下移植人肝癌裸鼠肿瘤生长的影响;运用免疫组化及Western blotting检测皮下移植人肝癌荷瘤鼠分子靶向治疗后肿瘤组织中Ll-cadherin、P53表达水平的变化以及相关信号通路的基因表达的变化,同时免疫组化和实时定量PCR观察联合分子靶向治疗的生物学安全性,排除脱靶效应。结果:1、皮下接种MHCC97H-mi-Ll-cadherin细胞组肿瘤与MHCC97H-mi-Luciferase以及未经处理的MHCC97H细胞组相比明显生长缓慢。皮下接种8周后,MHCC97H-miLuciferase荧光素酶干扰组和MHCC97H无处理对照组中全部动物肺部均见明显转移灶,而MHCC97H-miLl-cadherin靶基因干扰组中全部动物肺部均未见明显转移灶。2、经尾静脉注射靶基因干扰组MHCC97H-miLl-cadherin细胞与荧光素酶干扰组MHCC97H-miLuciferase细胞8周后,与皮下接种肿瘤转移情况一致,MHCC97H-miLuciferase荧光素酶干扰组中全部动物肺部均见明显转移灶,而MHCC97H-miLl-cadherin靶基因干扰组中全部动物肺部均未见明显转移灶。3、与TRIS缓冲生理盐水(TBS)组和(干扰荧光素酶的重组慢病毒)Lenti-miLuciferase组肿瘤相比,重组腺病毒携带的p53基因(rAd-p53)组肿瘤,Lenti-miLl-cadherin(干扰肝肠钙黏附蛋白的重组慢病毒)组肿瘤和Lenti-miLl-cadherin+rAd-p53组肿瘤生长可见明显抑制,其中Lenti-miLl-cadherin+rAd-p53联合分子靶向治疗组抑制最明显。4、Western blotting显示瘤内注射携带miR-30-based shRNA的慢病毒7周后,可明显抑制皮下移植肝癌MHCC97H中Ll-cadherin蛋白的表达,且相应对照组中均可见Ll-cadherin蛋白的高表达。TBS组,Lenti-miLuciferase组和rAd-p53组的β-Catenin蛋白高表达且表达水平相接近,而Lenti-miLl-cadherin组和Lenti-miLl-cadherin+rAd-p53组β-Catenin蛋白表达明显降低。rAd-p53组和Lenti-miLl-cadherin+rAd-p53组Bax的表达水平明显增高,Bcl-XL的表达水平相应降低。Lenti-miLl-cadherin组、rAd-p53组和Lenti-miLl-cadherin+rAd-p53组的caspase3活性蛋白的表达明显增高,尤其是Lenti-miLl-cadherin+rAd-p53联合治疗组的caspase3活性蛋白的表达最高。5、免疫组化和实时定量PCR未发现此联合分子靶向治疗的生物学安全性隐患和脱靶效应。结论:1、人源性高转移潜能肝癌细胞株MHCC97H中Ll-cadherin的表达被明显抑制后可导致肿瘤生长和转移能力的明显下降,为Ll-cadherin可能可以作为体内肝癌治疗的靶点提供理论依据。2、瘤内注射携带miR-30-based shRNA的慢病毒可显著抑制皮下移植肝癌MHCC97H中Ll-cadherin蛋白的表达,并抑制肿瘤组织的生长。3、瘤内注射携带miR-30-based shRNA的慢病毒联合rAd-p53分子靶向治疗,可更有效抑制肿瘤组织的生长,为肝癌的联合分子靶向治疗提供有效的实验依据。4、慢病毒载体Ll-cadherin基因干扰和抑癌基因P53联合分子靶向治疗中未发现明显生物学安全性隐患和脱靶效应。5、Ll-cadherin基因干扰和抑癌基因P53作用后相关信号通路研究提示Ll-cadherin的下调与GSK3β蛋白的激活和β-Catenin降解呈正相关。Lenti-miLl-cadherin和rAd-p53治疗肝癌分别通过不同途径调控肿瘤细胞凋亡,引起的肿瘤生长抑制,具有协同作用。更多还原

【Abstract】 PartⅠEstablishment,characterization and package of miR-30-based shRNA lentivirus targeted at LI-cadherinPurpose:The study designed the miR-30-based shRNA sequence targeted at LI-cadherin gene and packaged the recombinant lentivirus to study the role of LI-cadherin in the growth and metastasis of HCC.Materials and Methods:Based on miR-30-based shRNA library of Cold Spring Harbour carried by the PSM2 vector,the pLUNIG-miLI-cadherin as the recombinant working plasmid and pLUNIG-miLuciferase as non-specifically interfering control plasmid was established.Ca3(PO4) 2-DNA coprecipitates was used to transfect 293T cells with either the pLUNIG-miLI-cadherin or pLUNIG-miLuciferase and the other three helper plasmids PCMV-VSVG, PMDL-G/P-RRE and PRSV-REV.The virus supernatant was collected and condensed.Q-RT-PCR was used to examine the copies of lentivirus. MHCC97H cells were infected by the lentivirus.The stable transfectants were selected with G418 as titled MHCC97H-miLI-cadherin and MHCC97H-miLuciferase cell line.Q-RT-PCR,Immunocytochemistry and Western blotting were used to investigate whether the expression of LI-cadherin in MHCC97H-miLI-cadherin cells had been efficiently knocked down.Results:The sequences of the working plasmids were analyzed with BLAST method. Two working plasmids were acquired as titled pLUNIG-miLI-cadherin and pLUNIG-miLuciferase.The expression of LI-cadherin mRNA was downregulated to a certain degree.Immunocytochemistry showed the expression of LI-cadherin protein was significantly lower in MHCC97H-miLI-cadherin cells than in MHCC97H -miLuciferase cells.Western blotting further confirmed that the expression of LI-cadherin protein was significantly downregulated in MHCC97H-miLI-cadherin cells. The titre of the condensed lentivirus supernatant was above 10~9 as examined by Q-RT-PCR.The lentivirus was shown to have no function to replicate.Conclusion:The modified lentivirus is proved to be a novel generation with double selection system,which carries miR-30-based shRNA recombinant working plasmids targeted at LI-cadherin or Luciferase.Lentivirus has also been proved to be an ideal vector to induce shRNA into HCC cells.The expression of LI-cadherin protein in MHCC97H has been successfully knocked down with miR-30-based shRNA interfering method.A large quantity of package and production of the recombinant lentivirus with high titres has been successfully performed for the use in in vitro and in vivo experiments.PartⅡCharacterization of changes in biological properties of MHCC97H-miLI-cadherin cellsPurpose:To study the changes in the cell proliferation,adhesion,clone formation, migration,invasion,cell cycle,apoptosis,and related drugs sensitivity of MHCC97H-miLI-cadherin cells.To explore the possible underlying mechanisms.Materials and Methods:Cell proliferation and adhesion assays detected by comparing OD values. Cell cycle assays examined by PI/Rnase staining-Flow Cytometry.Cell apoptosis assays checked by TUNEL.Cloning formation assays measured by soft agar.Cell migration and invasion assays calculated by TRANSWELL methods.Drug sensitivity assays monitored by MTT.Signaling pathways exploration using Western blotting.Results:The cell growth and cell adhesion ability of MHCC97H-miLI-cadherin cells were markedly inhibited as compared to controls.Statistic analyses showed significant difference between MHCC97H-miLI-cadherin cells and controls.The percentage of cells in S phase in MHCC97H-miLI-cadherin cells was significantly less than that in MHCC97H-miLuciferase cells and blank control, whereas the percentage of cells in G0-G1 phase in MHCC97H-miLI-cadherin cells was significantly higher than that in MHCC97H-miLuciferase cells and blank control.The percentage of positive apoptotic cells in MHCC97H-miLI-cadherin cells was 8.52±1.43%,which was significantly higher than 1.55±0.33%in MHCC97H-miLuciferase cells.The number of clone formation,migration and invasion in MHCC97H-miLI-cadherin group was significantly less than that in MHCC97H-miLuciferase and blank control groups.The inhibition rate of MHCC97H-miLI-cadherin group was respectively 93.1%,90.2%and 88.3%. Statistic analysis showed significant difference between MHCC97H-miLI-cadherin group and control groups.The drug sensitivity and inhibition rate of Epirubcin,Carboplatin to MHCC97H-miLI-cadherin cells was significantly elevated as compared to MHCC97H-miLuciferase cells and blank control.Statistic analysis showed significant difference between MHCC97H-miLI-cadherin group and control groups.The drug sensitivity and inhibition rate of rAd-p53 to MHCC97H-miLI-cadherin cells was significantly elevated as compared to MHCC97H-miLuciferase cells and blank control.Statistic analysis showed significant difference between MHCC97H-miLI-cadherin group and control groups.The Wnt/β-catenin pathway was possibly involved in the biological changes of MHCC97H-miLI-cadherin cells.In brief,the downregulation of LI-cadherin activated GSK-3β,then led to the degradation ofβ-catenin,inhibited the expression of CyclinD1 and accordingly increased the expression of Rb.Conclusion:MHCC97H-miLI-cadherin cells show great changes in the cell biological properties as compared to MHCC97H-miLuciferase cells and blank control MHCC97H cells including retarded proliferation;decreased ability in cell adhesion,clone formation,cell migration and invasion;the decrease in the percentage of cell number in S phase and increase in the percentage of cell number in G0-G1 phase;the increase in the cell apoptosis and drug sensitivity.All these results demonstrate that the downregulation of LI-cadherin has obviously altered the tumor properties of the HCC cell line,strongly indicating the oncogene property of LI-cadherin.PartⅢInhibition of growth and metastasis of Hepatocellular Carcinoma by lentivirus-induced RNAi therapy combined with rAd-p53 gone therapy and involved signaling pathwaysPurpose:To study the changes in cancergenesis and metastasis of MHCC97H-miLI-cadherin cells after subcutaneous inoculation in nude mice. To study the changes in metastasis of MHCC97H-miLI-cadherin cells after tail vein injection in nude mice.To study the changes in tumor growth of subcutaneous inoculated HCC and the expression of LI-cadherin in tumor cells after combined gene therapies.To explore the signaling pathway related to LI-cadherin down-regulation.Materials and Methods:MHCC97H-miLI-cadherin cells,MHCC97H-miLuciferase cells,and blank control MHCC97H cells were subcutaneously inoculated into adult nude mice respectively to investigate the difference in cancergenesis and metastasis among the three groups.MHCC97H-miLI-cadherin cells, MHCC97H-miLuciferase cells,and blank control MHCC97H cells were injected into adult nude mice via tail vein respectively to investigate the difference in cancergenesis and metastasis among the three groups.The therapeutic effect of intratumor injection of lenti-miLI-Cadherin with and without rAd-p53 on the growth of subcutaneously inoculated HCC in nude mice was investigated.The expression of LI-cadherin and related molecules in subcutaneously inoculated HCC were investigated after intratumor injection of lenti-miLI-Cad with and without rAd-p53 by IHC and westen blotting assays. The biological safety and off-target effects of combined gene therapies were also investigated with IHC and Q-RT-PCR.Results:The subcutaneously inoculated tumor of MHCC97H-miLI-cadherin group grew much more slowly as compared to MHCC97H-miLuciferase and blank control groups.8 weeks after subcutaneous inoculation,all animals in MHCC97H-miLuciferase and blank control groups showed metastasis in lung, however,no metastasis was observed in MHCC97H-miLI-cadherin group. Similar to subcutaneous inoculation,all animals in MHCC97H-miLuciferase group via tail vein injection showed metastasis in lung and no metastasis was observed in MHCC97H-miLI-cadherin group.The growth of HCC tumor in rAd-p53 group,lenti-miLI-cadherin group and lenti-miLI-cadherin plus rAd-p53 group was remarkably inhibited as compared to TBS group and lenti-miLuciferase group.Among the former three groups, the growth of HCC tumor in lenti-miLI-cadherin plus rAd-p53 group showed the strongest inhibition.Western blotting showed that 4 weeks after the last intratumor injection of lenti-miLI-cadherin,the expression of LI-cadherin was significantly downregulated whereas high expression of LI-cadherin was shown in control groups.The expression of total GSK3βwere high and at the similar level in all five groups while the S9 phosphated GSK3βare apparently dawnregulated in lenti-miLI-cadherin group and lenti-miLI-cadherin plus rAd-p53 group.The expression ofβ-catenin in TBS group,lenti-miLuciferase group were high and at the similar level,which was in contrast to significant downregulation ofβ-catenin in lenti-miLI-cadherin group and lenti-miLI-cadherin plus rAd-p53 group.The expression of Cyclin D1 in TBS group,lenti-miLuciferase group was high and at the similar level,which was in contrast to significant downregulation of Cyclin D1 in lenti-miLI-cadherin group and lenti-miLI-cadherin plus rAd-p53 group and the expression of Rb were upregulated correspondingly.The expression of P53 were high in all five groups especially in rAd-p53 group and lenti-miLI-cadherin plus rAd-p53 group but without any obvious relationship with the LI-cadherin expression.The expression of Bax in rAd-p53 group and lenti-miLI-cadherin plus rAd-p53 group was elevated,whereas the expression of Bcl-XL decreased accordingly. The expression of cleaved caspase 3 in rAd-p53 group,lenti-miLI-cadherin group and lenti-miLI-cadherin plus rAd-p53 group was obviously induced.No risk in biological safety and off-target effects of combined gene therapies were observed using IHC and Q-RT-PCR assays.Conclusion:Downregulation of LI-cadherin expression in MHCC97H cells leads to the marked decrease in the tumor growth and metastasis,providing evidence that LI-cadherin can be therapeutic target for HCC treatment.Intratumor injection of lenti-miLI-cadherin can significantly downregulate the expression of LI-cadherin,which may be associated with inhibiting tumor growth.Intratumor injection of lenti-miLI-cadherin together with rAd-p53 shows greater effects on inhibiting tumor growth,providing evidence to apply combined gene therapies for treatment with HCC.No risk in biological safety and off-target effects of combined gene therapies have been observed during the period of drug admistration.The study of involved signaling pathway suggesting that the downregulation of LI-cadherin was closely related to activation of GSK-3βand degradation ofβ-catenin;indicating that the tumor growth inhibition due to lenti-miLI-cadherin and rAd-p53 therapy was associated with the different signaling pathway to cooperate and increase in the cell apoptosis.更多还原