肿瘤翻译调控蛋白（TCTP）对结肠癌细胞LoVo的生物学特性影响及安全型慢病毒载体构建探索
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摘要
大肠癌是世界范围内四大最常见恶性肿瘤之一,结直肠癌在西方国家占肿瘤死亡的第二位,全世界结直肠癌发病率以每年2%的速度上升,近年来我国的大肠癌的发病率呈上升趋势,且以每年4%的幅度递增,递增速度为世界水平的2倍。并且绝大多数的大肠癌患者死亡原因是由于转移,其中肝转移是结直肠癌主要的死亡原因之一,当结直肠癌确诊时,已经有20%—40%病例发生了肝转移,而治愈性根治术后再发的肝转移率为50%结直肠癌死亡的患者肝转移率高达45%-71%,
由于大肠癌的浸润转移是一个十分复杂、多步骤的过程,虽然有些大肠癌浸润转移的分子机制已有所阐明,但尚存在许多不确定的因素。尽管研究报道有百余种基因参与调控大肠癌的转移过程,这些基因为阐明转移的分子机制提供了重要的线索,但它们仍远远不能解释转移过程的复杂性和多样性。
目前,大肠癌的治疗以手术为主,辅以局部或全身放、化疗,但未能明显提高大肠癌的5年生存率,随着分子生物学及基因工程的发展,基因治疗已成为研究重点。
尽管有大量有关大肠癌基因治疗的深入研究,并已在临床取得一定疗效。但要真正达到大规模运用于临床治疗,还存在以下主要问题:①目前基因治疗的安全性没有很好解决,如对导入基因的控制、外缘基因及基因载体本身整合到宿主染色体带来的一系列问题等;②用于大肠癌理想载体的选择应具有安全性、高效性、靶向性、大容量性和可控性等特点,目前还难以找到可运用到理想的临床要求载体;③缺乏足够的基因治疗靶序列,大肠癌的发生是一种多基因水平调控的失控所致,单独基因治疗效果差。
由此进一步深入研究大肠癌的发病机制,寻找确定更多更有效的治疗靶基因,制备安全高效的基因治疗载体是大肠癌基因治疗中迫切需要解决的问题。本文第一部分着重研究肿瘤翻译调控蛋白(TCTP)对高转移性结肠癌细胞株的细胞生物学影响以及相关信号传导通路的研究。第二部分着重研究新型基因治疗用慢病毒载体生产体系的改进和优化。
第一部分通过构建靶向TCTP基因的shRNA质粒表达载体,下调TCTP在LoVo细胞中的表达。通过CCK-8实验检测LoVo细胞的增殖速度,粘附实验检测LoVo细胞对ECM的粘附能力,趋化实验和划痕实验检测LoVo细胞的运动能力,侵袭实验检测LoVo细胞对于ECM的分解穿透能力,EMSA以及荧光素酶实验检测TCTP下调后对于NF-κB活性的影响。通过成瘤实验检测TCTP下调对于LoVo细胞成瘤能力的影响,转移模型实验检测LoVo细胞体内肝转移能力的影响,通过2-D电泳和质谱分析鉴定与TCTP相关蛋白,并初步评价TCTP在正常人以及病人血清中的含量。
构建shRNA质粒表达载体,质粒酶切以及测序鉴定表明质粒构建成功。下调TCTP在LoVo细胞中的表达,经荧光显微镜观察各组LoVo细胞均出现绿色报告荧光,RT-PCR,western blotting,qRT-PCR分析鉴定TCTP的表达下调分别达到82%、89%、73%。
TCTP下调能够体外减慢LoVo细胞的增殖速度,shRNA-TCTP,shRNA-TCTP,LoVo parental组分别铺96孔板,每孔细胞数2000个。每株细胞5个复孔,铺7块板,连续测量7天。从第4天开始,shRNA-TCTP组生长速度明显慢于shncRNA-TCTP组和NC组,经过统计学分析后,shRNA—TCTP组在测量第1(F=1.773,P＞0.05)、2(F=0.048,P＞0.05)、3(F=7.865,P＞0.05)天时,shRNA-TCTP,shncRNA-TCTP,LoVo parental组三组细胞增殖曲线无显著性差异。第4(F=14.711,P＜0.001),5(F=64.453,P＜0.001),6(F=19.198,P＜0.001),7(F=56.696,P＜0.001)天时shRNA-TCTP增殖状态均较shncRNA-TCTP组或者LoVo parental减弱,有显著性差异。shRNA-TCTP组细胞增殖抑制率分别达到22.0%,32.8%,18.2%,15.1%(第4天—第7天数据分别进行方差分析后并进行两两比较,P＜0.05,经Bonferroni检验后,shRNA-TCTP组增殖状态均较shncRNA-TCTP或者LoVo parental减弱,并且有显著性差异,P＜0.001,shncRNA-TCTP LoVo与LoVo parental无显著性差异,P＞0.05)。
TCTP下调减弱LoVo细胞对ECM的粘附性,分别以基底膜主要成份Collagen I、FN、LN、VN为粘附介质进行LoVo shRNA-TCTP,shncRNA-TCTP细胞和LoVo parental的基底膜粘附实验,下调TCTP表达可分别抑制LoVo细胞对四种不同介质的粘附能力(LN F=42.80,P＜0.001;VN F=65.165,P＜0.001;FNF=319.345,P＜0.001;Collagen I F=161.39,P＜0.001以上四组检验中,经Bonferroni检验后,shRNA-TCTP组粘附能力均比shncRNA-TCTP或者LoVo parental组减弱,有显著性意义,P＜0.001,),但以LN为最明显,抑制率达59.6%,FN、VN、Collagen I分别为52.4%、51.2%、52.7%。TCTP缺失性功能实验从反方面证明TCTP对于大肠癌细胞的基底膜粘附性有重要影响。
TCTP下调减弱LoVo细胞对ECM的趋化运动性,待测细胞各组,每组设4个孔。取细胞对数生长期,用无血清RPMI1640培养过夜。Matrigel溶液均10μg/mL(1×PBS配制),每下室300μL,置37℃铺板1h。结果显示,shRNA-TCTPLoVo穿膜细胞数少于shncRNA-TCTP组和parental组,结果有统计学差异(方差分析后并进行两两比较。F=562.042,P＜0.001经Bonferroni检验后,shRNA-TCTP组穿膜细胞数均少于shncRNA-TCTP穿膜细胞数或者LoVoparental穿膜细胞数,差异有显著性意义,P＜0.001,shncRNA-TCTP LoVo穿膜细胞数与LoVo parental穿膜细胞数无显著性差异,P＞0.05)。划痕实验结果显示,TCTP表达下调明显延长LoVo细胞在matrigel胶上的愈合率。shRNA-TCTP在铺板12小时,24小时,36小时后的愈合率分别41.0±2.76%,65.3±2.2%,84.4±8.46%。明显低于同时间shncRNA-TCTP组的各个时间点愈合率62.7±2.2%,88.3±2.97%,98.4±2.17%以及LoVo parental组的各个时间点愈合率64.3±2.61%,88.5±4.19%,96.1±3.43%。经统计学分析在铺板12小时,24小时,36小时时测得的愈合率shRNA-TCTP均低于shncRNA-TCTP组和LoVo parental组,差异有显著性意义(12小时,F=79.573,P＜0.001;24小时,F=48.125,P＜0.001;36小时,F=5.737,P＜0.001,经Bonferroni检验后,shRNA-TCTP组均与shncRNA-TCTP或者LoVo parental均有明显差异,P＜0.001,shncRNA-TCTP LoVo组与LoVo parental组差异无显著性意义,P＞0.05)。
TCTP下调降低LoVo细胞对ECM的侵袭能力。粘附能力是肿瘤细胞发挥侵袭能力的基础,肿瘤细胞必须首先与细胞外基质发生粘附,然后才能通过肿瘤细胞本身或诱导宿主细胞分泌蛋白水解酶降解基质。为明确TCTP对大肠癌细胞侵袭能力的影响,在Transwell板的基础上,用Matrigel胶模仿基底膜构建侵袭性检测系统。实验结果从缺失性功能实验方面证明,抑制TCTP表达可以显著抑制LoVo细胞的侵袭能力(方差分析后并进行两两比较。F=71.512,P＜0.001经Bonferroni检验后,shRNA-TCTP组侵袭能力均比shncRNA-TCTP或者LoVoparental组侵袭能力减弱,差异有显著性意义,P＜0.001,shncRNA-TCTP组侵袭能力与LoVo parental侵袭能力差异无显著性意义,P＞0.05)。
TCTP表达下调明显抑制LoVo细胞成瘤能力,在裸鼠背部分别接种5×10~6的shRNA-TCTP LoVo,shncRNA-TCTP LoVo,LoVo parental细胞。三周后使用水合氯醛溶液麻醉小鼠后,将其断颈处死,取下瘤体,称重并经计算后得到shRNA-TCTP LoVo,shncRNA-TCTP LoVo,LoVo parental形成瘤体的平均重量分别为0.72±0.12g,2.44±0.21g,2.53±0.15g,统计结果表明shRNA-TCTP LoVo细胞形成瘤体重量与均轻于shncRNA-TCTP LoVo形成瘤体重量和LoVo细胞形成瘤体重量,差异有显著性意义(方差分析后并进行两两比较。F=161.3,P＜0.001经Bonferroni检验后,shRNA-TCTP LoVo细胞形成瘤体重量与均轻于shncRNA-TCTP LoVo形成瘤体重量和LoVo细胞形成瘤体重量,差异有显著性意义,P＜0.001,shncRNA-TCTPLoVo与LoVoparental无显著性差异,P＞0.05),然后迅速切成合适大小经4%多聚甲醛固定。并进行HE染色,HE染色,光镜下见癌细胞呈团块状分布,无腺腔样结构,胞浆丰富,核大不规则,核仁明显,可见多核瘤巨细胞,核分裂像易见,符合LoVo细胞形成的低分化腺癌的结构特征。
TCTP表达下调明显抑制LoVo细胞转移能力,小鼠接种初期活跃如常,体形无改变。3周后小鼠明显消瘦,腹部渐膨隆,行动渐迟缓,精神差,摄食量减少。于第三周末处死小鼠,解剖发现脾脏的接种部位均可见直径为0.3～0.5cm的灰白色结节,大小相对均一。腹腔内均有血性腹水,量不等。HE染色。虽然敲除TCTP表达未能阻止转移发生,但是LoVo shRNA-TCTP组肝脏表面结节数少于shncRNA-TCTP组肝脏表面结节数以及LoVo组肝脏表面结节数,差异有显著性意义(方差分析后并进行两两比较。F=277.736,P＜0.001经Bonferroni检验后,LoVo shRNA-TCTP组肝脏表面结节数少于shncRNA-TCTP组肝脏表面结节数以及LoVo组肝脏表面结节数,差异有显著性意义,P＜0.001,shncRNA-TCTP LoVo组与LoVo parental组肝脏表面结节数差异无显著性意义,P＞0.05)。.通过EMSA以及荧光素酶实验结果发现,TCTP表达下调抑制体内NF-κB的活性,核内p65含量降低。NF-κB是一类几乎存在于所有细胞内,能与多种基因的启动子或增强子NF-κB结合位点发生特异性结合并启动基因转录的一组转录调节因子。NF-κB是由亚单位P50和P65组成的异源二聚体,P50与DNA直接结合,P65具有转录活性。静息状态下,NF-κB和抑制性蛋白IκB-A结合滞留于细胞质中,在一定刺激作用下IKBα解离,NF-κB释放进入细胞核激活靶基因。质粒pNFκB-luc与海肾荧光素酶质粒载体分别共转然shRNA-TCTPLoVo,shncRNA-TCTP LoVo和LoVo parental细胞中,24h后,使用promega专用仪器检测萤火虫荧光素酶活性。其中海肾荧光素酶作为内参校正。由图可以观察到在shRNA-TCTP LoVo细胞中的NF-κB结合活性较shncRNA-TCTP和LoVo parental细胞中低,差异有显著性意义(方差分析后并进行两两比较。F=277.736,P＜0.001经Bonferroni检验后,shRNA-TCTP LoVo细胞中的NF-κB结合活性较shncRNA-TCTP和LoVo parental细胞中低,差异有显著性意义,P＜0.001,shncRNA-TCTP LoVo与LoVo parental中NF-κB活性差异无显著性意义),正常LoVo或者shncRNA-TCTP中的NF-κB活性约为shRNA-TCTP中的6倍。由EMSA图分析,shRNA-TCTP组中NF-κB结合活性明显降低。
经比较蛋白组学研究发现,TCTP表达下调引起细胞内27种蛋白质表达变化。其中24种蛋白表达水平上调,3种蛋白表达水平下调。并初步评价TCTP在正常人以及病人血清中的含量。这些蛋白大致分为泛素化系统组分(PSMA1,PSMA3,PSMA6,PSMC5,PSMD8,PSMD9,PSME3),肿瘤转移直接相关基因(HMGB1),细胞骨架相关蛋白(STMN1),离子通道相关蛋白(LASP1)等。
随机选取40个正常人和40个大肠癌病人,符合条件按方法中陈述。采用ELISA方法分别测定病人血清和正常人血清中抗TCTP抗体的含量。分别采用TRIFA竞争方法测定血清中TCTP的表达水平。由表1-4可以看到大肠癌病人血清中TCTP(t=40.894,P＜0.001)以及antiTCTP(t=-15.933,P＜0.001)抗体含量均与正常人值有显著性差异较高。其中有19例病人可以进行随访观察,并可以取得治疗半年左右的血清。对大肠癌病人治疗前后血清中TCTP以及antiTCTP的含量作配对t检验。发现大肠癌病人经手术,放疗,化疗治愈后,其中16对配对血清中的TCTP抗体以及抗原均有所下调(TCTP,t=26.964,P＜0.001;antiT℃TP,t=-63.327,P＜0.001)。3对配对血清中TCTP抗体以及抗原含量未有显著性变化,其中1例诊断为复发。
第二部分构建主框架质粒pVECRNA、包装质粒pGAGPOL及包膜质粒pVSVG。通过脂质体将这三个质粒共转染至BHK_(21)细胞,再用含有T_7RNA聚合酶基因的重组痘苗病毒vTF-3感染细胞,在生产细胞中,痘苗病毒转录翻译系统指导T_7RNA聚合酶的转录和翻译,然后T_7RNA聚合酶指导慢病毒载体cDNA的转录,痘苗病毒RNA聚合酶指导包装蛋白p24等以及包膜蛋白水泡性口炎病毒包膜蛋白(VSV-G)的转录翻译,最后由VSV-G包装慢病毒载体RNA以及功能蛋白形成慢病毒颗粒,经细胞分泌释放到培养上清中,收集培养上清经0.22μm滤膜过滤得到慢病毒载体。
RT-PCR及测序比对结果提示培养上清中含有慢病毒载体的基因组RNA。当三质粒与辅助痘苗病毒共转染生产细胞BHK_(21),48h后,共聚焦显微镜下观察到生产细胞表达p24蛋白,并且其主要分布于胞质中,而在细胞核中基本不表达,这提示质粒pGAGPOL构建成功;普通倒置荧光显微镜下观察到生产细胞表达GFP,提示质粒pVECRNA构建成功,以上结果也提示生产系统正常工作。将培养上清感染BHK_(21)细胞和293T细胞,48h后,倒置荧光显微镜下观察到BHK_(21)细胞和293T细胞均表达GFP,这提示利用本系统制备慢病毒载体颗粒的可行性。
为了提高系统产量,本研究对培养慢病毒载体所用的细胞系、质粒用量、重组痘苗病毒和细胞数比例(MOI)三个对慢病毒载体生产起关键作用的条件进行优化。每个因素取三个作用水平。所有结果在SPSS13.0软件中整理,按照3*3*3析因方差分析方法分析实验数据(计量资料用(?)±s表示,P＜0.05表示差异有显著性意义)。
实验结果表明细胞系、质粒用量以及MOI对载体产量有交互作用(F=7.728,P＜0.001)。
不同种生产细胞株之间慢病毒载体的产量有显著的统计学差异(F=1789.772、P＜0.001)。以BHK_(21)细胞的产量最高,载体拷贝数为(7036.78±3504.974)×10~8c/ml,大于Vero细胞产量(1670.89±968.55)×10~8c/ml及HepG_2产量(416.41±186.99)×10~8c/ml。
不同的MOI对病毒载体产量有显著统计学差异(F=311.543、P＜0.001)。其中病毒载体拷贝数在MOI为1时病毒载体拷贝数最高。
不同的质粒用量对病毒载体产量有显著性统计学差异(F=137.268、P＜0.001),病毒载体拷贝数在质粒用量为10μg条件下最高。
通过不同细胞株产量之间的比较,发现以BH_(21)为生产细胞时的系统产量远大于以HepG_2和Vero细胞为生产细胞时的系统产量。因此以BHK_(21)为生产细胞,对质粒用量和MOI进行3*3析因分析(计量资料用(?)±s表示,P＜0.05表示差异有显著性意义)。
通过结果可以得知:不同的质粒用量和MOI存在交互作用(F=9.980、p＜0.001)。
不同的质粒用量对病毒载体产量有显著性差异(F=92.997、P＜0.001)。其中病毒载体拷贝数在质粒用量为10μg时病毒载体拷贝数最高。采用Tamhane法(方差不齐)进行两两比较,发现质粒用量为5μg时的系统产量与质粒用量为10μg(P=0.031)和15μg(P=0.021)时系统产量均有显著性差异;质粒用量为10μg时的系统产量与质粒用量为15μg(P=0.987)时系统产量无显著性差异。
不同的MOI对病毒载体产量有显著性差异(F=174.778、P＜0.001),病毒拷贝数MOI为1条件下产量最高。采用Tamhane法(方差不齐)进行两两比较,得到MOI为0.1和1时,系统产量没有显著性差异(P=0.801),MOI为5时,系统产量与MOI为0.1(P=0.01)和1(P=0.01)时的系统产量均有显著性差异。
最后得出BHK_(21)为生产细胞,37℃,5%CO_2培养条件下,质粒用量为pVECRNA 10μg、pGAGPOL 10μg、pVSVG 2μg时,病毒载体拷贝数最高,达到(11707.67±802.263)×10~8c/ml。
在病毒载体拷贝数最优条件下GFP方法测得载体滴度达到(1.3±0.18)×10~8tu/ml。
总之,我们初步验证了TCTP下调对LoVo细胞生物学特性的影响,初步证实TCTP的下调能够抑制LoVo细胞体内体外增殖能力以及转移能力,提示TCTP有可能作为结肠癌基因治疗靶标,并且我们利用新的生产体系成功制备出慢病毒载体,为之后慢病毒载体生产体系的进一步完善以及临床基因治疗提供客观依据。
Colorectal cancer is one of the four most common worldwide malignancies,the incidence of colorectal cancer all over the world each year is rising at a rate of 2%in recent years,and the incidence of colorectal cancer in China is also rising,2 times of the world level.The vast majority cause of patients with colorectal cancer is the death induced by the liver metastases of colorectal cancer.
Because the invasion and metastasis of colorectal cancer is a very complex, multi-step process,although some invasion and metastasis mechanism on colorectal cancer has been clarified,there are still a lot of uncertainties.Researchers have reported that many genes involved in regulating the process of colorectal cancer has been identified,which provide an important clue to clarify the molecular mechanism, but they are still far from explaining the complexity and diversity progression of colorectal cancer.
At present,the treatment of colorectal cancer surgery,supplemented by local or systemic chemotherapy and radiotherapy failed to significantly improve colorectal cancer 5-year survival rate,with the molecular biology and the development of genetic engineering,gene therapy has been becoming a focus.
Even though there is a lot of in-depth studies on gene therapy for colorectal cancer,even there have been made in certain clinical efficacy,there are still the following questions:①the safety on gene therapy has not been resolved,such as genes transfected into the host chromosome bring a series of questions;②It is still difficult to find an ideal safety,efficiency,targeting,high-capacity vector applied to the clinical requirements;③more useful target genes are still urgently needed for effective treatment.
The first part of this article focuses on roles of tumor control protein translation (TCTP) in the metastasis ability of high-metastatic colon cancer cell lines LoVo.The second part focuses on improvement and optimization of a new type lentiviral vector production system.
The translationally controlled tumor protein(TCTP),a highly conserved protein, has been suggested as a tumor associated antigen.Its mRNA and protein expression levels tend to be higher in the colorectal cancers(CRC),compared to the corresponding normal tissue.Biological models of tumor reversion were established from human leukemia and breast cancer cell lines by using the H-1 parvovirus as a selective agent.Differential gene expression analysis was performed between the parental malignant cells and their revertants.TCTP was found to be the most strikingly down-regulated in tumor reversion.Furthermore,The levels of TCTP in the revertants from other three major solid cancers:colon,lung and melanoma cell lines have the same results,in addition,it was verified that inhibition of TCTP expression could induce changes in the malignant phenotype,when the v-src-transformed NIH3T3 cells transfected with antisense TCTP.The expression levels of TCTP were also found to be associated with Caco-2 differentiation by proteomics approach. Based on recently prognosis studies,TCTP has the potential to be identified as a new predictive marker for better understanding CRC progression involved in metastasis, however,the TCTP biological characteristics in colorectal cancer progression were still remained to be elucidated.Double-stranded RNA-mediated interference(RNAi) has recently emerged as a powerful genetic tool to silence gene expression in multiple organisms and widely used for gene function analysis.Small interfering RNA (siRNA) expression mediated by specific plasmids enables efficient and specific suppression of target gene expression in mammalian cells with maintenance of stable loss-of-function phenotypes.To investigate the biological role of TCTP in colorectal cancer progression,we used an RNAi-based proteomics approach with which we compared proteomes of LoVo cells before and after TCTP knockdown.LoVo initiated from a fragment of a metastatic tumor nodule in the left supraclavicular region of a male patient with a histologically proven diagnosis of colon adenocarcinoma.We investigated the phenotypic changes including the tumor proliferative,invasive and metastatic activities.Our results showed that knockdown of TCTP by shRNA inhibited proliferation,invasion and metastasis of LoVo cells both in vitro and in vivo.TCTP knockdown related proteins were identified by the comparative proteomes strategy.Several different spots were identified as components of the Ubiquitin-Proteasome System,proteins involved in the cytoskeleton biosynthesis and tumor metastasis,furthermore,gel electromobility shift assay(EMSA) and luciferase reporter assay revealed that down-regulation of TCTP might inhibit NF-κB binding activity in LoVo cells.These results imply that the expression of TCTP in CRC cell line LoVo is likely to partially associate with the ability of proliferation,migration and invasion,In addition,the NF-κB signaling pathway might be involved in these TCTP-related biological processes.
The inhibition efficiency of TCTP specific shRNA in LoVo cells were first examined by reverse transcription-PCR usingβ-actin as the internal reference. Western blotting also showed the reduced expression of TCTP protein in TCTP-shRNA transfected cells.TCTP and OAS1 mRNA levels were also measured by QRT-PCR.The TCTP mRNA levels were obviously reduced in the shRNA-TCTP LoVo cells;however,The IFN-response gene OAS1 mRNA levels have no significant changed in the three groups.
To investigate the effect of TCTP knockdown on functional alteration,growth of cells was firstly assessed by CCK-8 assay.As shown in Figure 1-6,compared with those TCTP-shncRNA transfected cells,the growth of TCTP-shRNA transfected cells was inhibited to 78.0%(P＜0.05),67.2%(P＜0.05),81.8%(P＜0.05) and 84.9% (P＜0.05) at 4th,5th,6th and 7th day,calculated by absorbance,respectively.
The effect of TCTP RNAi on motility of LoVo cells was examined using transwell(Costar,NY;pore size,8-mm) in 24-well dishes.Fewer TCTP-shRNA-transfected cells than TCTP-shncRNA-transfected and parental cells were observed when the polycarbonate filters were stained with crystal violet.The migration assay showed the number of cells invaded to the bottom chamber was much greater both in TCTP-shncRNA-transfected cells and in parental LoVo cells than in TCTP-shRNA-transfected cells,TCTP knockdown significantly inhibited the invasiveness of LoVo cells,by 87.5%.
To determine whether adhesion was affected by inhibiting expression of TCTP in LoVo cells,we tested attachment of LoVo cells to the extracellular matrix (fibronectin,vitronectin,laminin,and collagen I).Under identical experimental conditions,shRNA-TCTP plasmids transfected cells showed reduced adhesion to fibronectin,vitronectin,laminin and collagen I as compared to shncRNA-TCTP vector transfected cells.The result of adhesion assay demonstrated that TCTP silencing significantly inhibited the adhesive power of LoVo cells to laminin up to 59.6%.
Since knockdown of TCTP attenuated proliferation,migration,invasion activity of LoVo cells in vitro,we further investigated whether TCTP suppression would alter tumor growth and metastatic potential in nude mice.Cells(5×10~6/mouse) suspended in 0.2 ml DMEM were injected subcutaneously into the 6-week-old BALB/c nude mice at the right flanks.The animals were sacrificed on the 21st day after injection and the tumors were dissected and weighed.The tumorigenicity inhibition rate was up to 71.9%.shRNA-TCTP transfected,shncRNA-TCTP transfected and parental LoVo cells were injected into the spleen of nude mice.When the mice were sacrificed 5 weeks after injection,the number of the hepatic surface metastases of shRNA-TCTP group was significantly less than that of the tumors formed by shncRNA-TCTP transfected LoVo cells.These data indicated that suppressed TCTP expression in LoVo cells could inhibit tumor growth and metastasis in nude mice.
Histopathologic examinations revealed relatively normal liver tissue of the mice injected with TCTP-shRNA transfected cells and malignant liver tissue of the mice injected with TCTP-shncRNA transfected cells,there are more carcinoma cell colonies presented in liver samples from mice injected with shncRNA-TCTP transfected LoVo cells.
To explore the functional consequences of TCTP knockdown,we compared whole-cell proteomes on 2D-SDS-PAGE before and after knockdown of TCTP. Comparison of whole-cell proteomes yielded an alteration in 2D gel spots that were changed upon the removal of TCTP.These spots were excised from the gel,digested with trypsin,and analyzed by mass spectrometry.Information was gathered from 27 individual spots in total,from which 23 were down-regulated and 4 were up-regulated accompanied by the reduced levels of TCTP.The altered spots include Ubiquitin -Proteasome subunits,cancer proliferation,metastasis,cytoskeleton metabolism and ion binding related protein.To validate our mass spectrometry results,we obtained antibodies against a subset of the identified proteins and analyzed their expression levels in the shRNA-TCTP,shncRNA-TCTP and parental LoVo cells groups by Western blotting.We confirmed reduced expression of proteasome subunits of ubiquitin-proteasome system:PSMA1,PSMA3,PSME3,PSMA6,PSMD8 upon knockdown of TCTP;translational related protein HMGB1,the cell cytoskeleton metabolism related protein STMN1,LASP1 could also be confirmed.
The mechanism of TCTP-induced attenuated LoVo liver metastasis ability is not clearly understood.From the bioinformatics analysis on the proteomics results,some spots including proteasome subunits PSMA1,PSMA6,PSMD8, ubiquitin-proteasome system activator PSME3 and translationlly and related protein HMGB1 are closely involved in the NF-κB signaling pathways.The transcription factor nuclear factor-κB(NF-κB) family regulates the expression of genes involved in immune response,inflammation,cell proliferation,angiogenesis,metastasis,survival and suppression of apoptosis.To investigate whether NF-κB involved in the TCTP knockdown induced process,the shRNA-TCTP,shncRNA-TCTP cells and the parental LoVo were measured by the NF-κB luciferase reporter and EMSA assays.As shown in Figure.1-14,shRNA-TCTP plasmids transfected cells were associated with a six fold reduction of luciferase activity compared to negative control vector transfected cells,showing that knockdown TCTP inhibits the NF-κB binding activity. NF-κB activation was also examined by using electro mobility shift assays to measure binding of nucleus-located NF-κB to aγ-~(32)P labeled probe containing an NF-κB consensus sequence.As expected,nuclear extracts from shRNA-TCTP transfected cell populations showed the decreased binding activity to the labeled probe.There was no mobility-shifted band detected in no nucleus-extracted protein samples plus labeled probe as a control.To determine whether the probe bind specifically to NF-κB, we determined binding specificity by competition assays using the cold probe and the cold mutant probe.In the superfamily of Rel,p65 plays an important role in transcription after translocation to nuclei after phosphorylation and subsequent degradation of its inhibitor,IκBα.To study the nuclear transactivation of p65 in LoVo cells,we prepared nuclear extracts from three cells cultured for 24 h.A proper aliquot of protein from each cell lysate was loaded per lane onto a SDS-PAGE,transferred to polyvinyldifluoride membrane,and immunoblotted with anti-NF-κB,p65 specific antibodies.Results revealed that the enriching level of p65 was higher in nuclei of shncRNA-TCTP and parental LoVo cells than that in nuclei of shRNA-TCTP LoVo. The usual mechanism of NF-κB induction involves phosphorylation of IκBαby the IKK complex as a key step.The reverse results were detected for IκBαin cytoplasmic extracts compared to p65 in nuclear extracts.
Lentiviral vectors are attractive tools for human gene therapy.The goal of the present study was to develop an efficient transient-hybrid system for large-scale production of high titer lentiviral vector stocks.
In this system,BHK_(21) was co-transfected by three main plasmids consisting of transuding plasmid pVECRNA,packaging plasmid pgagpol,envelop plasmid pVSVG, and thereafter infected with the vaccinia vTF-3 containing bacteriophage T7 RNA polymerase gene using Lipofectamin2000~(TM).After 4 days,the culture supernatant of lentiviral vectors was collected and then followed by filtration through 0.22-um microporous membrane.
The culture supernatant was confirmed by the fragment of 96bp generated by RT-PCR and sequencing.After 48h,BHK_(21) was co-transfected by the three main plasmids and the helper virus.The expression of p24 was observed in BHK_(21) through indirect immunofluorescence by confocal microscopy,the p24 was mainly distributed in cytoplasm,rarely in cell nucleus.GFP was also expressed in BHK_(21) and 293T 48h later after they were infected with the culture supernatant.
3*3*3 factorial design was used to explore the yield optimization of this system. The type of cell lines,plasmids dosage and the MOI(the proportion between cell numbers and virus copies) were considered critical to the output of this system.The SPSS analysis results demonstrated that the three factors have significant effects on the output of the system respectively,and these three factors have significant interaction.
3*3 factorial design was used to explore the yield optimization of this system when BHK_(21) was utilized as the production cell line for that it was the best cell line for the LV production among the three mentioned cell lines containing BHK_(21),Vero, and HepG_2.The SPSS analysis results illustrated that the plasmids dosage and the MOI have significant effects on the system output respectively,and these two factors have significant interaction.
In short,the system yield archived to the favorable condition that BHK_(21) was co-transfected with 10μg pVECRNA,10μg pGAGPOL and 2μg pVSVG with the MOI 1 under the condition of 37℃and 5%CO_2.The LV copy number achieved to 1×10~(11)c/ml and the titers of LV stocks achieved to 1.3×10~8tu/ml.
In summary,the present study suggests that knockdown of TCTP in colon carcinoma LoVo cells might play at least a partial role in effectively inhibiting their migration,invasion in vitro and liver metastasis in vivo.the NF-κB signaling pathway might be involved in these TCTP-induced biological processes.Further work will be required to explain the concrete biological mechanism of TCTP on tumor growth and metastasis of colon adenocarcinoma LoVo cells.The LV has been successfully prepared with the new manufacturing system,which established a solid foundation for the optimization of further manufacture and clinical application.
由于大肠癌的浸润转移是一个十分复杂、多步骤的过程,虽然有些大肠癌浸润转移的分子机制已有所阐明,但尚存在许多不确定的因素。尽管研究报道有百余种基因参与调控大肠癌的转移过程,这些基因为阐明转移的分子机制提供了重要的线索,但它们仍远远不能解释转移过程的复杂性和多样性。
目前,大肠癌的治疗以手术为主,辅以局部或全身放、化疗,但未能明显提高大肠癌的5年生存率,随着分子生物学及基因工程的发展,基因治疗已成为研究重点。
尽管有大量有关大肠癌基因治疗的深入研究,并已在临床取得一定疗效。但要真正达到大规模运用于临床治疗,还存在以下主要问题:①目前基因治疗的安全性没有很好解决,如对导入基因的控制、外缘基因及基因载体本身整合到宿主染色体带来的一系列问题等;②用于大肠癌理想载体的选择应具有安全性、高效性、靶向性、大容量性和可控性等特点,目前还难以找到可运用到理想的临床要求载体;③缺乏足够的基因治疗靶序列,大肠癌的发生是一种多基因水平调控的失控所致,单独基因治疗效果差。
由此进一步深入研究大肠癌的发病机制,寻找确定更多更有效的治疗靶基因,制备安全高效的基因治疗载体是大肠癌基因治疗中迫切需要解决的问题。本文第一部分着重研究肿瘤翻译调控蛋白(TCTP)对高转移性结肠癌细胞株的细胞生物学影响以及相关信号传导通路的研究。第二部分着重研究新型基因治疗用慢病毒载体生产体系的改进和优化。
第一部分通过构建靶向TCTP基因的shRNA质粒表达载体,下调TCTP在LoVo细胞中的表达。通过CCK-8实验检测LoVo细胞的增殖速度,粘附实验检测LoVo细胞对ECM的粘附能力,趋化实验和划痕实验检测LoVo细胞的运动能力,侵袭实验检测LoVo细胞对于ECM的分解穿透能力,EMSA以及荧光素酶实验检测TCTP下调后对于NF-κB活性的影响。通过成瘤实验检测TCTP下调对于LoVo细胞成瘤能力的影响,转移模型实验检测LoVo细胞体内肝转移能力的影响,通过2-D电泳和质谱分析鉴定与TCTP相关蛋白,并初步评价TCTP在正常人以及病人血清中的含量。
构建shRNA质粒表达载体,质粒酶切以及测序鉴定表明质粒构建成功。下调TCTP在LoVo细胞中的表达,经荧光显微镜观察各组LoVo细胞均出现绿色报告荧光,RT-PCR,western blotting,qRT-PCR分析鉴定TCTP的表达下调分别达到82%、89%、73%。
TCTP下调能够体外减慢LoVo细胞的增殖速度,shRNA-TCTP,shRNA-TCTP,LoVo parental组分别铺96孔板,每孔细胞数2000个。每株细胞5个复孔,铺7块板,连续测量7天。从第4天开始,shRNA-TCTP组生长速度明显慢于shncRNA-TCTP组和NC组,经过统计学分析后,shRNA—TCTP组在测量第1(F=1.773,P＞0.05)、2(F=0.048,P＞0.05)、3(F=7.865,P＞0.05)天时,shRNA-TCTP,shncRNA-TCTP,LoVo parental组三组细胞增殖曲线无显著性差异。第4(F=14.711,P＜0.001),5(F=64.453,P＜0.001),6(F=19.198,P＜0.001),7(F=56.696,P＜0.001)天时shRNA-TCTP增殖状态均较shncRNA-TCTP组或者LoVo parental减弱,有显著性差异。shRNA-TCTP组细胞增殖抑制率分别达到22.0%,32.8%,18.2%,15.1%(第4天—第7天数据分别进行方差分析后并进行两两比较,P＜0.05,经Bonferroni检验后,shRNA-TCTP组增殖状态均较shncRNA-TCTP或者LoVo parental减弱,并且有显著性差异,P＜0.001,shncRNA-TCTP LoVo与LoVo parental无显著性差异,P＞0.05)。
TCTP下调减弱LoVo细胞对ECM的粘附性,分别以基底膜主要成份Collagen I、FN、LN、VN为粘附介质进行LoVo shRNA-TCTP,shncRNA-TCTP细胞和LoVo parental的基底膜粘附实验,下调TCTP表达可分别抑制LoVo细胞对四种不同介质的粘附能力(LN F=42.80,P＜0.001;VN F=65.165,P＜0.001;FNF=319.345,P＜0.001;Collagen I F=161.39,P＜0.001以上四组检验中,经Bonferroni检验后,shRNA-TCTP组粘附能力均比shncRNA-TCTP或者LoVo parental组减弱,有显著性意义,P＜0.001,),但以LN为最明显,抑制率达59.6%,FN、VN、Collagen I分别为52.4%、51.2%、52.7%。TCTP缺失性功能实验从反方面证明TCTP对于大肠癌细胞的基底膜粘附性有重要影响。
TCTP下调减弱LoVo细胞对ECM的趋化运动性,待测细胞各组,每组设4个孔。取细胞对数生长期,用无血清RPMI1640培养过夜。Matrigel溶液均10μg/mL(1×PBS配制),每下室300μL,置37℃铺板1h。结果显示,shRNA-TCTPLoVo穿膜细胞数少于shncRNA-TCTP组和parental组,结果有统计学差异(方差分析后并进行两两比较。F=562.042,P＜0.001经Bonferroni检验后,shRNA-TCTP组穿膜细胞数均少于shncRNA-TCTP穿膜细胞数或者LoVoparental穿膜细胞数,差异有显著性意义,P＜0.001,shncRNA-TCTP LoVo穿膜细胞数与LoVo parental穿膜细胞数无显著性差异,P＞0.05)。划痕实验结果显示,TCTP表达下调明显延长LoVo细胞在matrigel胶上的愈合率。shRNA-TCTP在铺板12小时,24小时,36小时后的愈合率分别41.0±2.76%,65.3±2.2%,84.4±8.46%。明显低于同时间shncRNA-TCTP组的各个时间点愈合率62.7±2.2%,88.3±2.97%,98.4±2.17%以及LoVo parental组的各个时间点愈合率64.3±2.61%,88.5±4.19%,96.1±3.43%。经统计学分析在铺板12小时,24小时,36小时时测得的愈合率shRNA-TCTP均低于shncRNA-TCTP组和LoVo parental组,差异有显著性意义(12小时,F=79.573,P＜0.001;24小时,F=48.125,P＜0.001;36小时,F=5.737,P＜0.001,经Bonferroni检验后,shRNA-TCTP组均与shncRNA-TCTP或者LoVo parental均有明显差异,P＜0.001,shncRNA-TCTP LoVo组与LoVo parental组差异无显著性意义,P＞0.05)。
TCTP下调降低LoVo细胞对ECM的侵袭能力。粘附能力是肿瘤细胞发挥侵袭能力的基础,肿瘤细胞必须首先与细胞外基质发生粘附,然后才能通过肿瘤细胞本身或诱导宿主细胞分泌蛋白水解酶降解基质。为明确TCTP对大肠癌细胞侵袭能力的影响,在Transwell板的基础上,用Matrigel胶模仿基底膜构建侵袭性检测系统。实验结果从缺失性功能实验方面证明,抑制TCTP表达可以显著抑制LoVo细胞的侵袭能力(方差分析后并进行两两比较。F=71.512,P＜0.001经Bonferroni检验后,shRNA-TCTP组侵袭能力均比shncRNA-TCTP或者LoVoparental组侵袭能力减弱,差异有显著性意义,P＜0.001,shncRNA-TCTP组侵袭能力与LoVo parental侵袭能力差异无显著性意义,P＞0.05)。
TCTP表达下调明显抑制LoVo细胞成瘤能力,在裸鼠背部分别接种5×10~6的shRNA-TCTP LoVo,shncRNA-TCTP LoVo,LoVo parental细胞。三周后使用水合氯醛溶液麻醉小鼠后,将其断颈处死,取下瘤体,称重并经计算后得到shRNA-TCTP LoVo,shncRNA-TCTP LoVo,LoVo parental形成瘤体的平均重量分别为0.72±0.12g,2.44±0.21g,2.53±0.15g,统计结果表明shRNA-TCTP LoVo细胞形成瘤体重量与均轻于shncRNA-TCTP LoVo形成瘤体重量和LoVo细胞形成瘤体重量,差异有显著性意义(方差分析后并进行两两比较。F=161.3,P＜0.001经Bonferroni检验后,shRNA-TCTP LoVo细胞形成瘤体重量与均轻于shncRNA-TCTP LoVo形成瘤体重量和LoVo细胞形成瘤体重量,差异有显著性意义,P＜0.001,shncRNA-TCTPLoVo与LoVoparental无显著性差异,P＞0.05),然后迅速切成合适大小经4%多聚甲醛固定。并进行HE染色,HE染色,光镜下见癌细胞呈团块状分布,无腺腔样结构,胞浆丰富,核大不规则,核仁明显,可见多核瘤巨细胞,核分裂像易见,符合LoVo细胞形成的低分化腺癌的结构特征。
TCTP表达下调明显抑制LoVo细胞转移能力,小鼠接种初期活跃如常,体形无改变。3周后小鼠明显消瘦,腹部渐膨隆,行动渐迟缓,精神差,摄食量减少。于第三周末处死小鼠,解剖发现脾脏的接种部位均可见直径为0.3～0.5cm的灰白色结节,大小相对均一。腹腔内均有血性腹水,量不等。HE染色。虽然敲除TCTP表达未能阻止转移发生,但是LoVo shRNA-TCTP组肝脏表面结节数少于shncRNA-TCTP组肝脏表面结节数以及LoVo组肝脏表面结节数,差异有显著性意义(方差分析后并进行两两比较。F=277.736,P＜0.001经Bonferroni检验后,LoVo shRNA-TCTP组肝脏表面结节数少于shncRNA-TCTP组肝脏表面结节数以及LoVo组肝脏表面结节数,差异有显著性意义,P＜0.001,shncRNA-TCTP LoVo组与LoVo parental组肝脏表面结节数差异无显著性意义,P＞0.05)。.通过EMSA以及荧光素酶实验结果发现,TCTP表达下调抑制体内NF-κB的活性,核内p65含量降低。NF-κB是一类几乎存在于所有细胞内,能与多种基因的启动子或增强子NF-κB结合位点发生特异性结合并启动基因转录的一组转录调节因子。NF-κB是由亚单位P50和P65组成的异源二聚体,P50与DNA直接结合,P65具有转录活性。静息状态下,NF-κB和抑制性蛋白IκB-A结合滞留于细胞质中,在一定刺激作用下IKBα解离,NF-κB释放进入细胞核激活靶基因。质粒pNFκB-luc与海肾荧光素酶质粒载体分别共转然shRNA-TCTPLoVo,shncRNA-TCTP LoVo和LoVo parental细胞中,24h后,使用promega专用仪器检测萤火虫荧光素酶活性。其中海肾荧光素酶作为内参校正。由图可以观察到在shRNA-TCTP LoVo细胞中的NF-κB结合活性较shncRNA-TCTP和LoVo parental细胞中低,差异有显著性意义(方差分析后并进行两两比较。F=277.736,P＜0.001经Bonferroni检验后,shRNA-TCTP LoVo细胞中的NF-κB结合活性较shncRNA-TCTP和LoVo parental细胞中低,差异有显著性意义,P＜0.001,shncRNA-TCTP LoVo与LoVo parental中NF-κB活性差异无显著性意义),正常LoVo或者shncRNA-TCTP中的NF-κB活性约为shRNA-TCTP中的6倍。由EMSA图分析,shRNA-TCTP组中NF-κB结合活性明显降低。
经比较蛋白组学研究发现,TCTP表达下调引起细胞内27种蛋白质表达变化。其中24种蛋白表达水平上调,3种蛋白表达水平下调。并初步评价TCTP在正常人以及病人血清中的含量。这些蛋白大致分为泛素化系统组分(PSMA1,PSMA3,PSMA6,PSMC5,PSMD8,PSMD9,PSME3),肿瘤转移直接相关基因(HMGB1),细胞骨架相关蛋白(STMN1),离子通道相关蛋白(LASP1)等。
随机选取40个正常人和40个大肠癌病人,符合条件按方法中陈述。采用ELISA方法分别测定病人血清和正常人血清中抗TCTP抗体的含量。分别采用TRIFA竞争方法测定血清中TCTP的表达水平。由表1-4可以看到大肠癌病人血清中TCTP(t=40.894,P＜0.001)以及antiTCTP(t=-15.933,P＜0.001)抗体含量均与正常人值有显著性差异较高。其中有19例病人可以进行随访观察,并可以取得治疗半年左右的血清。对大肠癌病人治疗前后血清中TCTP以及antiTCTP的含量作配对t检验。发现大肠癌病人经手术,放疗,化疗治愈后,其中16对配对血清中的TCTP抗体以及抗原均有所下调(TCTP,t=26.964,P＜0.001;antiT℃TP,t=-63.327,P＜0.001)。3对配对血清中TCTP抗体以及抗原含量未有显著性变化,其中1例诊断为复发。
第二部分构建主框架质粒pVECRNA、包装质粒pGAGPOL及包膜质粒pVSVG。通过脂质体将这三个质粒共转染至BHK_(21)细胞,再用含有T_7RNA聚合酶基因的重组痘苗病毒vTF-3感染细胞,在生产细胞中,痘苗病毒转录翻译系统指导T_7RNA聚合酶的转录和翻译,然后T_7RNA聚合酶指导慢病毒载体cDNA的转录,痘苗病毒RNA聚合酶指导包装蛋白p24等以及包膜蛋白水泡性口炎病毒包膜蛋白(VSV-G)的转录翻译,最后由VSV-G包装慢病毒载体RNA以及功能蛋白形成慢病毒颗粒,经细胞分泌释放到培养上清中,收集培养上清经0.22μm滤膜过滤得到慢病毒载体。
RT-PCR及测序比对结果提示培养上清中含有慢病毒载体的基因组RNA。当三质粒与辅助痘苗病毒共转染生产细胞BHK_(21),48h后,共聚焦显微镜下观察到生产细胞表达p24蛋白,并且其主要分布于胞质中,而在细胞核中基本不表达,这提示质粒pGAGPOL构建成功;普通倒置荧光显微镜下观察到生产细胞表达GFP,提示质粒pVECRNA构建成功,以上结果也提示生产系统正常工作。将培养上清感染BHK_(21)细胞和293T细胞,48h后,倒置荧光显微镜下观察到BHK_(21)细胞和293T细胞均表达GFP,这提示利用本系统制备慢病毒载体颗粒的可行性。
为了提高系统产量,本研究对培养慢病毒载体所用的细胞系、质粒用量、重组痘苗病毒和细胞数比例(MOI)三个对慢病毒载体生产起关键作用的条件进行优化。每个因素取三个作用水平。所有结果在SPSS13.0软件中整理,按照3*3*3析因方差分析方法分析实验数据(计量资料用(?)±s表示,P＜0.05表示差异有显著性意义)。
实验结果表明细胞系、质粒用量以及MOI对载体产量有交互作用(F=7.728,P＜0.001)。
不同种生产细胞株之间慢病毒载体的产量有显著的统计学差异(F=1789.772、P＜0.001)。以BHK_(21)细胞的产量最高,载体拷贝数为(7036.78±3504.974)×10~8c/ml,大于Vero细胞产量(1670.89±968.55)×10~8c/ml及HepG_2产量(416.41±186.99)×10~8c/ml。
不同的MOI对病毒载体产量有显著统计学差异(F=311.543、P＜0.001)。其中病毒载体拷贝数在MOI为1时病毒载体拷贝数最高。
不同的质粒用量对病毒载体产量有显著性统计学差异(F=137.268、P＜0.001),病毒载体拷贝数在质粒用量为10μg条件下最高。
通过不同细胞株产量之间的比较,发现以BH_(21)为生产细胞时的系统产量远大于以HepG_2和Vero细胞为生产细胞时的系统产量。因此以BHK_(21)为生产细胞,对质粒用量和MOI进行3*3析因分析(计量资料用(?)±s表示,P＜0.05表示差异有显著性意义)。
通过结果可以得知:不同的质粒用量和MOI存在交互作用(F=9.980、p＜0.001)。
不同的质粒用量对病毒载体产量有显著性差异(F=92.997、P＜0.001)。其中病毒载体拷贝数在质粒用量为10μg时病毒载体拷贝数最高。采用Tamhane法(方差不齐)进行两两比较,发现质粒用量为5μg时的系统产量与质粒用量为10μg(P=0.031)和15μg(P=0.021)时系统产量均有显著性差异;质粒用量为10μg时的系统产量与质粒用量为15μg(P=0.987)时系统产量无显著性差异。
不同的MOI对病毒载体产量有显著性差异(F=174.778、P＜0.001),病毒拷贝数MOI为1条件下产量最高。采用Tamhane法(方差不齐)进行两两比较,得到MOI为0.1和1时,系统产量没有显著性差异(P=0.801),MOI为5时,系统产量与MOI为0.1(P=0.01)和1(P=0.01)时的系统产量均有显著性差异。
最后得出BHK_(21)为生产细胞,37℃,5%CO_2培养条件下,质粒用量为pVECRNA 10μg、pGAGPOL 10μg、pVSVG 2μg时,病毒载体拷贝数最高,达到(11707.67±802.263)×10~8c/ml。
在病毒载体拷贝数最优条件下GFP方法测得载体滴度达到(1.3±0.18)×10~8tu/ml。
总之,我们初步验证了TCTP下调对LoVo细胞生物学特性的影响,初步证实TCTP的下调能够抑制LoVo细胞体内体外增殖能力以及转移能力,提示TCTP有可能作为结肠癌基因治疗靶标,并且我们利用新的生产体系成功制备出慢病毒载体,为之后慢病毒载体生产体系的进一步完善以及临床基因治疗提供客观依据。
Colorectal cancer is one of the four most common worldwide malignancies,the incidence of colorectal cancer all over the world each year is rising at a rate of 2%in recent years,and the incidence of colorectal cancer in China is also rising,2 times of the world level.The vast majority cause of patients with colorectal cancer is the death induced by the liver metastases of colorectal cancer.
Because the invasion and metastasis of colorectal cancer is a very complex, multi-step process,although some invasion and metastasis mechanism on colorectal cancer has been clarified,there are still a lot of uncertainties.Researchers have reported that many genes involved in regulating the process of colorectal cancer has been identified,which provide an important clue to clarify the molecular mechanism, but they are still far from explaining the complexity and diversity progression of colorectal cancer.
At present,the treatment of colorectal cancer surgery,supplemented by local or systemic chemotherapy and radiotherapy failed to significantly improve colorectal cancer 5-year survival rate,with the molecular biology and the development of genetic engineering,gene therapy has been becoming a focus.
Even though there is a lot of in-depth studies on gene therapy for colorectal cancer,even there have been made in certain clinical efficacy,there are still the following questions:①the safety on gene therapy has not been resolved,such as genes transfected into the host chromosome bring a series of questions;②It is still difficult to find an ideal safety,efficiency,targeting,high-capacity vector applied to the clinical requirements;③more useful target genes are still urgently needed for effective treatment.
The first part of this article focuses on roles of tumor control protein translation (TCTP) in the metastasis ability of high-metastatic colon cancer cell lines LoVo.The second part focuses on improvement and optimization of a new type lentiviral vector production system.
The translationally controlled tumor protein(TCTP),a highly conserved protein, has been suggested as a tumor associated antigen.Its mRNA and protein expression levels tend to be higher in the colorectal cancers(CRC),compared to the corresponding normal tissue.Biological models of tumor reversion were established from human leukemia and breast cancer cell lines by using the H-1 parvovirus as a selective agent.Differential gene expression analysis was performed between the parental malignant cells and their revertants.TCTP was found to be the most strikingly down-regulated in tumor reversion.Furthermore,The levels of TCTP in the revertants from other three major solid cancers:colon,lung and melanoma cell lines have the same results,in addition,it was verified that inhibition of TCTP expression could induce changes in the malignant phenotype,when the v-src-transformed NIH3T3 cells transfected with antisense TCTP.The expression levels of TCTP were also found to be associated with Caco-2 differentiation by proteomics approach. Based on recently prognosis studies,TCTP has the potential to be identified as a new predictive marker for better understanding CRC progression involved in metastasis, however,the TCTP biological characteristics in colorectal cancer progression were still remained to be elucidated.Double-stranded RNA-mediated interference(RNAi) has recently emerged as a powerful genetic tool to silence gene expression in multiple organisms and widely used for gene function analysis.Small interfering RNA (siRNA) expression mediated by specific plasmids enables efficient and specific suppression of target gene expression in mammalian cells with maintenance of stable loss-of-function phenotypes.To investigate the biological role of TCTP in colorectal cancer progression,we used an RNAi-based proteomics approach with which we compared proteomes of LoVo cells before and after TCTP knockdown.LoVo initiated from a fragment of a metastatic tumor nodule in the left supraclavicular region of a male patient with a histologically proven diagnosis of colon adenocarcinoma.We investigated the phenotypic changes including the tumor proliferative,invasive and metastatic activities.Our results showed that knockdown of TCTP by shRNA inhibited proliferation,invasion and metastasis of LoVo cells both in vitro and in vivo.TCTP knockdown related proteins were identified by the comparative proteomes strategy.Several different spots were identified as components of the Ubiquitin-Proteasome System,proteins involved in the cytoskeleton biosynthesis and tumor metastasis,furthermore,gel electromobility shift assay(EMSA) and luciferase reporter assay revealed that down-regulation of TCTP might inhibit NF-κB binding activity in LoVo cells.These results imply that the expression of TCTP in CRC cell line LoVo is likely to partially associate with the ability of proliferation,migration and invasion,In addition,the NF-κB signaling pathway might be involved in these TCTP-related biological processes.
The inhibition efficiency of TCTP specific shRNA in LoVo cells were first examined by reverse transcription-PCR usingβ-actin as the internal reference. Western blotting also showed the reduced expression of TCTP protein in TCTP-shRNA transfected cells.TCTP and OAS1 mRNA levels were also measured by QRT-PCR.The TCTP mRNA levels were obviously reduced in the shRNA-TCTP LoVo cells;however,The IFN-response gene OAS1 mRNA levels have no significant changed in the three groups.
To investigate the effect of TCTP knockdown on functional alteration,growth of cells was firstly assessed by CCK-8 assay.As shown in Figure 1-6,compared with those TCTP-shncRNA transfected cells,the growth of TCTP-shRNA transfected cells was inhibited to 78.0%(P＜0.05),67.2%(P＜0.05),81.8%(P＜0.05) and 84.9% (P＜0.05) at 4th,5th,6th and 7th day,calculated by absorbance,respectively.
The effect of TCTP RNAi on motility of LoVo cells was examined using transwell(Costar,NY;pore size,8-mm) in 24-well dishes.Fewer TCTP-shRNA-transfected cells than TCTP-shncRNA-transfected and parental cells were observed when the polycarbonate filters were stained with crystal violet.The migration assay showed the number of cells invaded to the bottom chamber was much greater both in TCTP-shncRNA-transfected cells and in parental LoVo cells than in TCTP-shRNA-transfected cells,TCTP knockdown significantly inhibited the invasiveness of LoVo cells,by 87.5%.
To determine whether adhesion was affected by inhibiting expression of TCTP in LoVo cells,we tested attachment of LoVo cells to the extracellular matrix (fibronectin,vitronectin,laminin,and collagen I).Under identical experimental conditions,shRNA-TCTP plasmids transfected cells showed reduced adhesion to fibronectin,vitronectin,laminin and collagen I as compared to shncRNA-TCTP vector transfected cells.The result of adhesion assay demonstrated that TCTP silencing significantly inhibited the adhesive power of LoVo cells to laminin up to 59.6%.
Since knockdown of TCTP attenuated proliferation,migration,invasion activity of LoVo cells in vitro,we further investigated whether TCTP suppression would alter tumor growth and metastatic potential in nude mice.Cells(5×10~6/mouse) suspended in 0.2 ml DMEM were injected subcutaneously into the 6-week-old BALB/c nude mice at the right flanks.The animals were sacrificed on the 21st day after injection and the tumors were dissected and weighed.The tumorigenicity inhibition rate was up to 71.9%.shRNA-TCTP transfected,shncRNA-TCTP transfected and parental LoVo cells were injected into the spleen of nude mice.When the mice were sacrificed 5 weeks after injection,the number of the hepatic surface metastases of shRNA-TCTP group was significantly less than that of the tumors formed by shncRNA-TCTP transfected LoVo cells.These data indicated that suppressed TCTP expression in LoVo cells could inhibit tumor growth and metastasis in nude mice.
Histopathologic examinations revealed relatively normal liver tissue of the mice injected with TCTP-shRNA transfected cells and malignant liver tissue of the mice injected with TCTP-shncRNA transfected cells,there are more carcinoma cell colonies presented in liver samples from mice injected with shncRNA-TCTP transfected LoVo cells.
To explore the functional consequences of TCTP knockdown,we compared whole-cell proteomes on 2D-SDS-PAGE before and after knockdown of TCTP. Comparison of whole-cell proteomes yielded an alteration in 2D gel spots that were changed upon the removal of TCTP.These spots were excised from the gel,digested with trypsin,and analyzed by mass spectrometry.Information was gathered from 27 individual spots in total,from which 23 were down-regulated and 4 were up-regulated accompanied by the reduced levels of TCTP.The altered spots include Ubiquitin -Proteasome subunits,cancer proliferation,metastasis,cytoskeleton metabolism and ion binding related protein.To validate our mass spectrometry results,we obtained antibodies against a subset of the identified proteins and analyzed their expression levels in the shRNA-TCTP,shncRNA-TCTP and parental LoVo cells groups by Western blotting.We confirmed reduced expression of proteasome subunits of ubiquitin-proteasome system:PSMA1,PSMA3,PSME3,PSMA6,PSMD8 upon knockdown of TCTP;translational related protein HMGB1,the cell cytoskeleton metabolism related protein STMN1,LASP1 could also be confirmed.
The mechanism of TCTP-induced attenuated LoVo liver metastasis ability is not clearly understood.From the bioinformatics analysis on the proteomics results,some spots including proteasome subunits PSMA1,PSMA6,PSMD8, ubiquitin-proteasome system activator PSME3 and translationlly and related protein HMGB1 are closely involved in the NF-κB signaling pathways.The transcription factor nuclear factor-κB(NF-κB) family regulates the expression of genes involved in immune response,inflammation,cell proliferation,angiogenesis,metastasis,survival and suppression of apoptosis.To investigate whether NF-κB involved in the TCTP knockdown induced process,the shRNA-TCTP,shncRNA-TCTP cells and the parental LoVo were measured by the NF-κB luciferase reporter and EMSA assays.As shown in Figure.1-14,shRNA-TCTP plasmids transfected cells were associated with a six fold reduction of luciferase activity compared to negative control vector transfected cells,showing that knockdown TCTP inhibits the NF-κB binding activity. NF-κB activation was also examined by using electro mobility shift assays to measure binding of nucleus-located NF-κB to aγ-~(32)P labeled probe containing an NF-κB consensus sequence.As expected,nuclear extracts from shRNA-TCTP transfected cell populations showed the decreased binding activity to the labeled probe.There was no mobility-shifted band detected in no nucleus-extracted protein samples plus labeled probe as a control.To determine whether the probe bind specifically to NF-κB, we determined binding specificity by competition assays using the cold probe and the cold mutant probe.In the superfamily of Rel,p65 plays an important role in transcription after translocation to nuclei after phosphorylation and subsequent degradation of its inhibitor,IκBα.To study the nuclear transactivation of p65 in LoVo cells,we prepared nuclear extracts from three cells cultured for 24 h.A proper aliquot of protein from each cell lysate was loaded per lane onto a SDS-PAGE,transferred to polyvinyldifluoride membrane,and immunoblotted with anti-NF-κB,p65 specific antibodies.Results revealed that the enriching level of p65 was higher in nuclei of shncRNA-TCTP and parental LoVo cells than that in nuclei of shRNA-TCTP LoVo. The usual mechanism of NF-κB induction involves phosphorylation of IκBαby the IKK complex as a key step.The reverse results were detected for IκBαin cytoplasmic extracts compared to p65 in nuclear extracts.
Lentiviral vectors are attractive tools for human gene therapy.The goal of the present study was to develop an efficient transient-hybrid system for large-scale production of high titer lentiviral vector stocks.
In this system,BHK_(21) was co-transfected by three main plasmids consisting of transuding plasmid pVECRNA,packaging plasmid pgagpol,envelop plasmid pVSVG, and thereafter infected with the vaccinia vTF-3 containing bacteriophage T7 RNA polymerase gene using Lipofectamin2000~(TM).After 4 days,the culture supernatant of lentiviral vectors was collected and then followed by filtration through 0.22-um microporous membrane.
The culture supernatant was confirmed by the fragment of 96bp generated by RT-PCR and sequencing.After 48h,BHK_(21) was co-transfected by the three main plasmids and the helper virus.The expression of p24 was observed in BHK_(21) through indirect immunofluorescence by confocal microscopy,the p24 was mainly distributed in cytoplasm,rarely in cell nucleus.GFP was also expressed in BHK_(21) and 293T 48h later after they were infected with the culture supernatant.
3*3*3 factorial design was used to explore the yield optimization of this system. The type of cell lines,plasmids dosage and the MOI(the proportion between cell numbers and virus copies) were considered critical to the output of this system.The SPSS analysis results demonstrated that the three factors have significant effects on the output of the system respectively,and these three factors have significant interaction.
3*3 factorial design was used to explore the yield optimization of this system when BHK_(21) was utilized as the production cell line for that it was the best cell line for the LV production among the three mentioned cell lines containing BHK_(21),Vero, and HepG_2.The SPSS analysis results illustrated that the plasmids dosage and the MOI have significant effects on the system output respectively,and these two factors have significant interaction.
In short,the system yield archived to the favorable condition that BHK_(21) was co-transfected with 10μg pVECRNA,10μg pGAGPOL and 2μg pVSVG with the MOI 1 under the condition of 37℃and 5%CO_2.The LV copy number achieved to 1×10~(11)c/ml and the titers of LV stocks achieved to 1.3×10~8tu/ml.
In summary,the present study suggests that knockdown of TCTP in colon carcinoma LoVo cells might play at least a partial role in effectively inhibiting their migration,invasion in vitro and liver metastasis in vivo.the NF-κB signaling pathway might be involved in these TCTP-induced biological processes.Further work will be required to explain the concrete biological mechanism of TCTP on tumor growth and metastasis of colon adenocarcinoma LoVo cells.The LV has been successfully prepared with the new manufacturing system,which established a solid foundation for the optimization of further manufacture and clinical application.
引文
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