食管癌EC9706细胞Livin和MTA1双基因沉默的实验研究
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摘要
背景和目的:
Livin是新近发现的凋亡抑制蛋白家族(inhibitor of apoptosis protein,IAP)成员之一,可以和半胱氨酸天冬氨酸特异性蛋白酶(Caspase)直接结合起到凋亡抑制作用。Livin主要在胎盘组织中表达,在胎儿发育过程的多种组织中其表达量较高,而在正常人终末分化组织中呈低表达或不表达。研究发现在多数肿瘤组织中存在Livin高表达现象;它的过度表达可引起凋亡不足而与肿瘤发生密切相关;同时在肿瘤的化疗中,IAP的表达可以有效介导肿瘤的凋亡抵抗。近年来对Livin的研究表明,Livin有可能作为诱导肿瘤凋亡治疗的一个新靶点而倍受关注。
肿瘤转移相关基因(metastasis associated gene,MTA)是一个不断快速增长的基因家族。现已发现该基因包含3类(MTA1,MTA2,MTA3)及6个亚型(MTA1,MTA1s,MTA-ZG29p,MTA2,MTA3,MTA3L)。MTA1被认为是细胞核重构和脱乙酰基复合物(nucleosome remodeling and histone deacetylase,NuRD)的组成部分之一,它通过影响染色质的状态来调整转录复制过程,调控组蛋白脱乙酰基从而发挥其生物学作用。目前多数研究认为MTA1的高表达与一些上皮源性肿瘤的侵袭转移密切相关。MTA1可能是通过参与信号传导与基因表达调控一系列有关浸涧转移的蛋白而起重要作用。MTA1基因的作用将为恶性肿瘤侵袭转移的调控提供一个有效的作用靶点和控制手段。
在基因治疗方面,RNA干扰(RNA interference,RNAi)是一个崭新的技术,它是由双链RNA引发的转录后基因沉默(posttranscriptional gene silencing,PTGS)现象。该技术能够有效使转录后基因沉默,甚至可以代替基因敲除,为高效特异地阻断致病基因表达提供了强有力的手段。已经有许多研究者们通过RNAi技术来干扰某些致病基因的表达使肿瘤的生物学行为发生一定程度的逆转。但肿瘤的发生与致病都是一个多因素和多基因参与的过程,往往只依靠解决单一的一种异常分子往往不能够完全达到预期的目的。鉴于凋亡抑制基因和肿瘤转移基因均是肿瘤发生以及肿瘤致死的主要因素,已经有学者利用RNAi技术沉默MTA1或Livin,并且取得了一定的肿瘤的抑制效果。有关MTA1和Livin基因在食管鳞癌组织中的表达以及在EC9706细胞诱导Livin和MTA1双基因沉默的实验研究,迄今国内外均未见报道。因此,本研究拟通过探讨:食管癌组织中Livin和MTA1基因的表达情况,及其与浸润转移的关系;构建Livin和MTA1双基因沉默慢病毒型siRNA载体,建立Livin和MTA1双基因沉默食管癌细胞模型;研究Livin和MTA1双基因沉默对食管癌细胞生长、凋亡、侵袭、转移等方面的作用;进而为食管癌靶向治疗提供理论和实验基础。本研究共分以下四个部分。
第一部分:Livin、MTA1基因在食管鳞癌组织中的表达分析
方法:
收集45例食管癌手术标本,每例标本分别取癌灶及远端5cm处正常粘膜组织(即远癌正常组织)两部分。采用实时荧光定量逆转录多聚酶链式反应(Real-time RT-PCR)分别检测食管癌组织和对应正常粘膜组织中Livinα、Livinβ和MTA1 mRNA的表达;同时采用免疫组化方法分析食管癌组织和对应正常粘膜组织中Livin和MTA1蛋白的表达。
结果:
1.45例食管癌组织中Livinα和Livinβ的mRNA相对表达量显著高于对应远癌正常组织中Livinα和Livinβ的mRNA相对表达量(P＜0.01);食管癌组织中LivinβmRNA平均表达量高于LivinαmRNA,但差异无显著性(P＞0.05)。
2.45例食管癌组织中Livin蛋白表达阳性率为95.6%(43/45),对应远癌正常组织中Livin蛋白表达阳性率仅为6.7%(3/45),二者差异有高度显著性(P＜0.01)。
3.45例食管癌组织中MTA1 mRNA相对表达量显著高于对应远癌正常组织中MTA1 mRNA的相对表达量(P＜0.01);有淋巴结转移标本中MTA1mRNA表达量显著高于无淋巴结转移标本,差异有高度显著性(P＜0.01)。
4.45例食管癌组织中MTA1蛋白表达阳性率为55.6%(25/45),对应远癌正常组织中却未见MTA1蛋白表达阳性表达。18例伴有淋巴结转移食管癌组织中MTA1蛋白的阳性表达率为100%(18/18);而无淋巴结转移27例食管癌组织中MTA1蛋白的阳性率为25.9%(7/27),两组问差别有显著性(P＜0.05)。
第二部分:构建针对MTA1和Livin基因的siRNA载体及其沉默效果的分析
方法:
利用Takara、Ambion和Promega siRNA靶序列分析设计系统,扫描人MTA1 cDNA编码序列(NM-004689)和Livin cDNA编码序列(NM_139317),依据siRNA靶序列设计原则,经BLAST同源性分析,最终MTA1基因确定5个靶序列,Livin基因最终确定2个靶序列,同时随机组合出一条无关对照siRNA序列。分别合成8对编码短发卡RNA序列的DNA单链,两端分别加入BamHI和XhoⅠ的酶切残基。将合成的8对发卡DNA分别退火,分别克隆入siRNA载体(pRNAT-U6.2/Lenti),通过PCR筛选和DNA序列分析,得到重组子pRNAT-U6.2/Lenti-M286、pRNAT-U6.2/Lenti-M481、pRNAT-U6.2/Lenti-M533、pRNAT-U6.2/Lenti-M1018、pRNAT-U6.2/Lenti-M1332、pRNAT-U6.2/Lenti-L440、pRNAT-U6.2/Lenti-L652和pRNAT-U6.2/Lenti-Con。与辅助包装质粒共转染293FT细胞,进行病毒包装;收集、纯化得到重组慢病毒颗粒。分别感染食管癌EC9706细胞,G418筛选得到稳定感染细胞株。分别用荧光定量RT-PCR和Western blot检测MTA1或Livin基因表达沉默效果。
结果:
1.筛选得到5个针对MTA1基因的siRNA靶序列,286-304nt,481-499 nt,533-551 nt,1018-1036 nt,1332-1350 nt;2个针对Livin基因的siRNA靶序列,分别为440-458 nt和652-470 nt。
2.成功构建5个针对MTA1基因、2个针对Livin基因和1个无关序列慢病毒siRNA表达载体(pRNAT-U6.2/Lenti-M286、pRNAT-U6.2/Lenti-M481、pRNAT-U62/Lenti-M533、pRNAT-U6.2/Lenti-M1018、pRNAT-U62/Lenti-M1332、pRNAT-U62/Lenti-L440、pRNAT-U6.2/Lenti-L652和pRNAT-U6.2/Lenti-Con),包装纯化得到重组慢病毒颗粒。
3.经过G418筛选感染细胞,得到5株稳定感染沉默MTA1表达的EC9706细胞株(siM286、siM481、siM533、siM1018和siM1332),得到2株稳定感染沉默Livin表达的EC9706细胞株(siL440和siL652)。
4.感染靶向MTA1基因重组siRNA慢病毒的实验组食管癌EC9706细胞中MTA1 mRNA和蛋白表达都受到抑制,但抑制程度不同。其中pRNAT-U6.2/Lenti-M481包装产生的慢病毒,感染EC9706细胞的抑制效果最佳,MTA1mRNA和蛋白表达几乎完全抑制。
5.感染靶向Livin基因重组siRNA慢病毒的实验组食管癌EC9706细胞中LivinmRNA和蛋白表达都受到抑制。pRNAT-U6.2/Lenti-L440包装产生的慢病毒,感染EC9706细胞的抑制效果强于pRNAT-U6.2/Lenti-L652,Livin表达几乎完全抑制。
第三部分:RNAi干扰MTA1和Livin基因表达对食管癌EC9706细胞凋亡和侵袭迁移的影响
方法:
选用第二部分已构建出最有效沉默MTA1和Livin基因的siRNA载体,XhoⅠ和PmeⅠ双酶切pRNAT-U6.2/Lenti-Livin,回收XhoⅠ粘端和PmeⅠ平端线形化载体;AccⅡ和XhoⅠ酶切pRNAT-U6.2/Lenti-MTA1,回收475bp的MTA1siRNA单元:将MTA1 siRNA单元克隆入线形化pRNAT-U6.2/Lenti-Livin,经PCR筛选鉴定得到Livin和MTA1双基因沉默siRNA载体(pRNAT-U6.2/Lenti-Livin-MTA1)。与辅助包装质粒共转染293FT细胞,进行病毒包装;收集、纯化得到重组Livin和MTA1双基因沉默慢病毒颗粒。感染食管癌EC9706细胞,G418筛选得到稳定感染Livin和MTA1双基因沉默细胞株;荧光定量RT-PCR和Western-blot检测其沉默效果。对单一Livin基因沉默细胞株、单一MTA1基因沉默细胞株和Livin、MTA1双基因沉默细胞株,分别测定细胞生长曲线,Caspase-3活性,检测细胞凋亡和放射线敏感性的影响;测定细胞侵袭迁移能力。
结果:
1.构建出针对Livin和MTA1双基因沉默慢病毒siRNA载体pRNAT-U6.2/Lenti-Livin-MTA1,包装出Livin和MTA1双基因沉默慢病毒颗粒。
2.筛选得到稳定Livin和MTA1双基因沉默的食管癌细胞株。
3.Livin和MTA1双基因沉默慢病毒感染食管癌EC9706细胞,能够有效抑制细胞Livin和MTA1基因mRNA和蛋白的表达,与单一沉默Livin基因和MTA1基因的抑制效果相当。
4.单一Livin基因沉默和Livin、MTA1双基因沉默,可使食管癌EC9706细胞生长速度显著减缓,凋亡率、Caspase-3活性和放射线敏感性显著增加,差异均有显著性(P＜0.05)。
5.单一MTA1基因沉默和Livin、MTA1双基因沉默,可使食管癌EC9706细胞划痕愈合减缓和穿透胶能力降低。并且,Livin、MTA1双基因沉默细胞穿透胶能力降低的效果显著优于单一MTA1基因沉默细胞。
第四部分:Livin和MTA1双基因沉默EC9706细胞裸鼠移植瘤的实验研究
方法:
用第三部分建立的Livin和MTA1双基因沉默EC9706细胞株作为实验组,以第二部分建立的无关siRNA对照食管癌EC9706细胞株作为无关siRNA对照组,用未感染的食管癌EC9706细胞株作为空白对照组。培养上述3种细胞,分别接种5周龄的雌性BALB/c裸鼠。测量肿瘤大小绘制肿瘤生长曲线,28天后处死裸鼠,取肿瘤组织称重。荧光定量RT-PCR检测移植瘤组织中Livin、MTA1和PCNA mRNA的表达;免疫组化法检测移植瘤组织中Livin、MTA1蛋白的表达;流式细胞术检测移植瘤细胞的凋亡情况。
结果:
1.MTA1和Livin双基因沉默的食管癌EC9706细胞移植瘤,瘤体生长速度减慢。
2.MTA1和Livin双基因沉默的食管癌EC9706细胞移植瘤平均重量为228.1mg,两个对照组分别为1265.6mg和1341.8mg,组间比差异有显著性(P＜0.05)。
3.MTA1和Livin双基因沉默的食管癌EC9706细胞移植瘤细胞中PCNAmRNA表达量比两个对照组PCNA mRNA表达量显著降低,差异有显著性(P＜0.05)。
4.MTA1和Livin双基因沉默的食管癌EC9706细胞移植瘤细胞凋亡率为12.75%,而2个对照组细胞凋亡率分别为1.53%和1.68%,差异有显著性(P＜0.05)。
结论:
1.食管癌组织中存在MTA1和Livin基因呈现高表达;MTA1基因表达与食管癌淋巴结转移密切相关。MTA1蛋白表达水平可能成为判断食管癌淋巴结转移的重要指标。
2.成功构建出高效Livin和MTA1双基因沉默的慢病毒siRNA载体。
3.筛选得到稳定Livin和MTA1双基因沉默的食管癌EC9706细胞株:Livin、MTA1单一基因沉默的食管癌EC9706细胞株。
4.体外细胞实验证实,Livin和MTA1双基因沉默,可使食管癌EC9706细胞生长速度显著减缓,浸润和侵袭能力显著降低,肿瘤细胞凋亡率和放射线敏感性显著增加,并且效果优于单一基因沉默。
5.裸鼠体内实验证实,沉默食管癌EC9706细胞MTA1和Livin双基因的表达后,可以有效的抑制移植瘤的生长,并且增加了肿瘤细胞凋亡。
6.同时沉默Livin和MTA1基因的表达,有望成为食管癌靶向基因治疗的新方法。
Background and Aim
Livin was one member of IAP(inhibitor of apoptosis protein) family which can directly combine with Caspase(cysteine-containing aspartate-specific proteases), to play a role in antiapoptosis.The Livin expression in the placenta tissue and its elevated level in the embryonic developing tissues are found;while there is no detectable expression of Livin in the normal adult terminal differentiated tissue.The overexpression of Livin in most tumors can cause cells to resist apoptosis,which is closely correlated to tumorigenesis and tumor progression.With more and more studies Livin may become a novel molecule target for anti-apoptosis in tumor therapy.
Metastasis associated gene(MTA) is a gene family,including three kinds (MTA1,MTA2,MTA3)and six subtypes(MTA1,MTA1s,MTA-ZG29p,MTA2, MTA3,MTA3L).The MTA1 functions as a nucleosome remodeling and histone deacetylase(NuRD) component with nucleosome reconstitution and histone deacetylation enzyme activity.These characteristics can modulate gene transcription and replication procedures through ATP-dependent NuRD acetylation and deacetylation to affect the chromatin state,which may contribute to modulating the protein expression related to tumor metastasis and invasion via signal transduction and gene expression regulation.A lot of studies showed that the enhanced expression level of MTA1 had close relationship to cancer invasion and metastasis.The MTA1 gene may become an effective target for modulating malignant tumor invasion and metastasis.
With the development of gene therapy,RNA interference(RNAi) technique emerged.It is a phenomenon of post-transcriptional gene silencing with double stranded RNA,which causes effective silencing of homologous mRNA molecules, and also provides an effective way to inhibit the pathogenesis gene expression.It has been confirmed that some tumor biological behaviors could be reversed by interfering pathogenesis gene expression.However,multiple factors and genes may participate in tumorigenesis process,so that the expecting effect cannot be always carried out by interfering single molecule expression.Combination of Livin and MTA1 may be a significant factor for interfering tumorigenesis and malignant invasion,and some researchers have silenced them by RNAi,which exhibited some inhibiting effects on tumor.The aims of this study:①To detect Livin and MTA1 expression in the esophageal tumor tissue and to reveal the two molecules' relationship to tumor metastasis and invasion;②To construct silenced siRNA Lentivirus vectors targeting Livin and MTA1 and to construct the esophageal tumor cell models with silenced Livin and MTA1 genes.③To examine the cell growth, apoptosis,metastasis and invasion of the esophageal tumor cells after silencing Livin and MTA1 genes,and to provide some theoretical and experimental data to support targeting therapy of esophageal carcinoma.
PartⅠ:Analysis of the Livin and MTA1 Expressions in the Esophageal Carcinoma
Methods:
45 samples of esophageal tumor tissues were collected,the tumor focus and the normal mucous membrane tissues(near distal end 5cm) were obtained from each sample.The mRNA expression level of Livinα,Livinβand MTA1were detected by real-time RT-PCR,the protein expressions of Livin and MTA1 were assayed by immunohistochemistry.
Results:
1.The mRNA expression level of Livinαand Livinβdetected by RT-PCR in 45 samples of the esophageal tumor tissue was higher than that of the corresponding normal tissue,5 cm far from the tumors(P＜0.01);the expression level average of Livinβwas higher than that of Livinα,but there was no significant difference between them(P＞0.05).
2.The positive rate of Livin protein expression examined by immunohistochemistry was 95.6%(43/45) in 45 samples of the esophageal tumor tissue,while the positive rate of Livin protein expression was only 6.7%(3/45 ) in the corresponding normal tissue.There was significant difference between them(P＜0.01).
3.The mRNA expression level of MTA1 detected by RT-PCR in 45 samples of the esophageal tumor tissue was higher than that of the corresponding normal tissue (P＜0.01);the mRNA expression level of MTA1 in the samples with lymph node metastasis was remarkably higher than that of the samples without lymph node metastasis.There was significant difference between them(P＜0.01).
4.The positive rate of MTA1 protein expression examined by immuno -histochemistry was 55.6%(25/45) in 45 samples of the esophageal tumor tissue, but MTA1 protein expression was not found in the corresponding normal tissue. The positive rate of MTA1 protein expression was 100%(18/18) in 18 samples of the esophageal tumor tissue with lymph node metastasis,but it was 25.9% (7/27) in 27 samples without lymph node metastasis.There was significant difference between them(P＜0.05).
PartⅡ:Construction of siRNA Vectors Targeting Respective Livin and MTA1 Genes and Detection of their Silencing Effects
Methods:
According to the principle of designing siRNA sequence,the Takara,Ambion and Promega siRNA sequence for designing and analyzing software system was used to scan human MTA1 cDNA sequence(NM_004689) and Livin cDNA sequence(NM_139317).Five siRNA sequences targeting MTA1 and two siRNA sequences targeting Livin were analyzed by BLAST homology and a contrast sequence(Con) was randomly assorted.Eight DNA oligonucleotides of short hairpin RNA sequence were synthesized and annealed into double strands respectively with BamHI and XhoI restricted incision enzyme sites;eight hairpin DNAs were cloned into siRNA expression vector(pRNAT-U6.2/Lenti) that had been cut by BamHI and XhoI restriction enzymes;the siRNA expression vector (pRNAT-U6.2/Lenti-M286,pRNAT-U6.2-/Lenti-M481,pRNAT-U6.2/Lenti-M533, pRNAT-U6.2/Lenti-M1018,pRNAT-U6.2/Lenti-M1332,pRNAT-U6.2/Lenti-L440, pRNAT-U6.2/Lenti-L652 and pRNAT-U6.2/Lenti-Con) were obtained by PCR and DNA sequencing,which were transfected into 293FT cells with aiding packaging plasmid.The Lentivirus particles were obtained by packaging,collecting and purifying;The esophageal carcinoma cell line EC9706 were infected respectively by these Lentivirus particles,the infected stable cell lines were established by G418 screening;The silencing effect was detected by real time RT- PCR and Western blotting.
Results:
1.Five siRNA sequences targeting MTA1 were obtained(286-304nt,481-499nt, 533-551nt,1018-1036nt,1332-1350nt);Two siRNA sequences targeting Livin were obtained(440-458nt and 652-670nt).
2.Five Lentivirus siRNA expression vectors targeting MTA1(pRNAT-U6.2/Lenti -M286,pRNAT-U6.2/Lenti-M481,pRNAT-U6.2/Lenti-M533,pRNAT-U6.2/ Lenti-M1018,pRNAT-U6.2/Lenti-M1332),two Lentivirus siRNA vectors targeting Livin(pRNAT-U6.2/Lenti-L440 and pRNAT-U6.2/Lenti-L652) and a contrast Lentivirus siRNA expression vector(pRNAT-U6.2/Lenti-Con) were successfully constructed,and the recombinated Lentivirus particles were obtained by packaging and purifying.
3.Five infected stable EC9706 cell lines silencing MTA1(siM286,siM481,siM533, siM1018 and siM1332 ) and two infected stable EC9706 cell lines silencing Livin (siL440 and siL652 ) were establisked via G418 screening.
4.The mRNA and protein expressions of MTA1 in cells of each group transfected by the Lentivirus vectors targeting MTA1 were obviously inhibited,but their silencing effects were different;among them,the silencing effect of pRNAT-U6.2/Lenti-M481 was the best,they were about completely depressed.
5.The mRNA and protein expression of Livin in cells of each group transfected by the Lentivirus vectors targeting Livin were markedly inhibited,and the silenced effect of pRNAT-U6.2/Lenti-L440 was better than that of pRNAT-U6.2/Lenti -L652,they were about completely depressed.
PartⅢ:Effects on Apoptosis and Migration of Esophageal EC9706 Cancer Cells Silenced by Reconstructed Single siRNA Vector Targeting both Livin and MTA1 Genes
Methods:
Based on the siRNA vectors constructed and screened in the second part,the putative most effective siRNA vector,recombinant single siRNA vector targeting both Livin and MTA1 genes was constructed.The pRNAT-U6.2/Lenti-Livin was cut by XhoI and PmeI,then the lineafizated vector was reclaimed;pRNAT-U6.2/Lenti -MTA1 was cut by AccII and XhoI;MTA1 siRNA unit with 475bp was reclaimed and cloned into the lineafizated pRNAT-U6.2/Lenti-Livin.The pRNAT-U6.2-Livin-MTA1 was identified and obtained by PCR,which was transfected into 293FT cells with aiding packaging plasmid.The Lentivirus particles silencing both Livin and MTA1 were packaged,collected and purified.The EC9706 cells were transfected by these Lentivirus particles.The infected stable cell lines were established via G418 screening respectively.The silencing effect detected by real time RT-PCR and Western blotting;the apoptosis effect detected by flow cytometry,the growth curve and sensitivity to radioactive ray detected by MTT assay,the migration ability detected by Matrigel Invasion Assay and the Caspase-3 activity detected by related kit were examined in respective established stable cell lines,targeting single Livin,targeting single MTA1 and targeting double Livin and MTA1.
Results:
1.The Lentivirus vector targeting both Livin and MTA1(pRNAT-U6.2/Lenti -Livin-MTA1) was successfully constructed;the Lentivirus particles silencing Livin and MTA1 were packaged.
2.The stable esophageal carcinoma cell line silencing both Livin and MTA1 genes was obtained by screening.
3.Both mRNA and protein expressions of Livin and MTA1 genes were inhibited in the EC9706 cells transfected by the Lentivirus particles silencing both Livin and MTA1 genes,and the silencing effect was similar to that of silencing Livin or MTA1 alone.
4.The cell growth velocity was reduced,while the apoptosis rate,the Caspase-3 activity and the sensitivity to radioactive ray obviously was increased in the EC9706 cell lines,containing silencing single Livin and silencing both Livin and MTA1.There was significant difference between the experimental group and the control group(P＜0.05).
5.The Matrigel invasion ability repressed in the EC9706 cell lines,silencing single MTA1 and silencing both Livin and MTA1.Besides,the repressing effect of Matrigel invasion ability in the cells silencing both Livin and MTA1 was better than that of the cells silencing MTA1 alone.
PartⅣ:The Nude Mouse Transplanted Tumor with EC9706 Cells Silencing both MTA1 and Livin Genes
Methods:
The EC9706 cell line silencing both MTA1 and Livin,the irrelevant siRNA EC9706 cell line and the untransfected EC9706 cell line were considered as the experimental group,the irrelevant siRNA control group and the untransfected control group respectively.Three above-mentioned cell lines were cultivated and inoculated into the female BALB/c nude mice aged 5 weeks old.The tumor growth curve was determined by measuring the tumor size;the nude mice were killed 28 days later and the tumors were weighted.The mRNA and protein expressions of Livin,MTA1 and PCNA were detected by real time PCR and immunohistochemistry;the apoptosis was detected by flow cytometry.
Results:
1.The tumor growth speed was retarded in the transplanted tumor with EC9706 cell line silencing both MTA1 and Livin.
2.The average weight of the transplanted tumor with EC9706 cell line silencing both MTA1 and Livin was 228.1 mg,the average weights were 1265.6 mg and 1341.8 mg in the other two control groups respectively.There was significant difference between them(P＜0.05).
3.The mRNA expression level of PCNA in the transplanted tumor with EC9706 cell line silencing both MTA1 and Livin was lower than that of the two control groups.There was significant difference between them(P＜0.05).
4.The cell apoptosis rate was 12.75%in the transplanted tumor with EC9706 cell line silencing both MTA1 and Livin;the cell apoptosis rates were 1.53%and 1.68%respectively in the other two control groups.There was significant difference between them(P＜0.05).
Conclusions
1.There are elevated expression level of Livin gene and MTA1 gene in the esophageal carcinoma tissue;and the MTA1 expression level is closely related to lymph node metastasis of the esophageal carcinoma.The protein expression level of MTA1 may be an important marker for evaluating lymph node metastasis of the esophageal carcinoma.
2.The effective Lentivirus vector silencing both Livin and MTA1 genes was constructed successfully.
3.The stable EC9706 cell lines silencing both Livin and MTA1 genes,silencing single Livin gene and silencing single MTA1 gene were established respectively.
4.The experiment in vitro indicates that silencing both Livin and MTA1 genes can reduce the cancer cell growth and invasion capacity;while apoptosis and sensitivity to radioactive ray markedly enhance,its effect is better than that of silencing Livin or MTA1 gene alone.
5.The experiment on nude mice exhibits that silencing both Livin and MTA1 genes can inhibit growth of transplanted tumor and facilitate the tumor apoptosis.
6.To silence Livin and MTA1 may provide a novel strategy for target gene therapy of the esophageal carcinoma.
Livin是新近发现的凋亡抑制蛋白家族(inhibitor of apoptosis protein,IAP)成员之一,可以和半胱氨酸天冬氨酸特异性蛋白酶(Caspase)直接结合起到凋亡抑制作用。Livin主要在胎盘组织中表达,在胎儿发育过程的多种组织中其表达量较高,而在正常人终末分化组织中呈低表达或不表达。研究发现在多数肿瘤组织中存在Livin高表达现象;它的过度表达可引起凋亡不足而与肿瘤发生密切相关;同时在肿瘤的化疗中,IAP的表达可以有效介导肿瘤的凋亡抵抗。近年来对Livin的研究表明,Livin有可能作为诱导肿瘤凋亡治疗的一个新靶点而倍受关注。
肿瘤转移相关基因(metastasis associated gene,MTA)是一个不断快速增长的基因家族。现已发现该基因包含3类(MTA1,MTA2,MTA3)及6个亚型(MTA1,MTA1s,MTA-ZG29p,MTA2,MTA3,MTA3L)。MTA1被认为是细胞核重构和脱乙酰基复合物(nucleosome remodeling and histone deacetylase,NuRD)的组成部分之一,它通过影响染色质的状态来调整转录复制过程,调控组蛋白脱乙酰基从而发挥其生物学作用。目前多数研究认为MTA1的高表达与一些上皮源性肿瘤的侵袭转移密切相关。MTA1可能是通过参与信号传导与基因表达调控一系列有关浸涧转移的蛋白而起重要作用。MTA1基因的作用将为恶性肿瘤侵袭转移的调控提供一个有效的作用靶点和控制手段。
在基因治疗方面,RNA干扰(RNA interference,RNAi)是一个崭新的技术,它是由双链RNA引发的转录后基因沉默(posttranscriptional gene silencing,PTGS)现象。该技术能够有效使转录后基因沉默,甚至可以代替基因敲除,为高效特异地阻断致病基因表达提供了强有力的手段。已经有许多研究者们通过RNAi技术来干扰某些致病基因的表达使肿瘤的生物学行为发生一定程度的逆转。但肿瘤的发生与致病都是一个多因素和多基因参与的过程,往往只依靠解决单一的一种异常分子往往不能够完全达到预期的目的。鉴于凋亡抑制基因和肿瘤转移基因均是肿瘤发生以及肿瘤致死的主要因素,已经有学者利用RNAi技术沉默MTA1或Livin,并且取得了一定的肿瘤的抑制效果。有关MTA1和Livin基因在食管鳞癌组织中的表达以及在EC9706细胞诱导Livin和MTA1双基因沉默的实验研究,迄今国内外均未见报道。因此,本研究拟通过探讨:食管癌组织中Livin和MTA1基因的表达情况,及其与浸润转移的关系;构建Livin和MTA1双基因沉默慢病毒型siRNA载体,建立Livin和MTA1双基因沉默食管癌细胞模型;研究Livin和MTA1双基因沉默对食管癌细胞生长、凋亡、侵袭、转移等方面的作用;进而为食管癌靶向治疗提供理论和实验基础。本研究共分以下四个部分。
第一部分:Livin、MTA1基因在食管鳞癌组织中的表达分析
方法:
收集45例食管癌手术标本,每例标本分别取癌灶及远端5cm处正常粘膜组织(即远癌正常组织)两部分。采用实时荧光定量逆转录多聚酶链式反应(Real-time RT-PCR)分别检测食管癌组织和对应正常粘膜组织中Livinα、Livinβ和MTA1 mRNA的表达;同时采用免疫组化方法分析食管癌组织和对应正常粘膜组织中Livin和MTA1蛋白的表达。
结果:
1.45例食管癌组织中Livinα和Livinβ的mRNA相对表达量显著高于对应远癌正常组织中Livinα和Livinβ的mRNA相对表达量(P＜0.01);食管癌组织中LivinβmRNA平均表达量高于LivinαmRNA,但差异无显著性(P＞0.05)。
2.45例食管癌组织中Livin蛋白表达阳性率为95.6%(43/45),对应远癌正常组织中Livin蛋白表达阳性率仅为6.7%(3/45),二者差异有高度显著性(P＜0.01)。
3.45例食管癌组织中MTA1 mRNA相对表达量显著高于对应远癌正常组织中MTA1 mRNA的相对表达量(P＜0.01);有淋巴结转移标本中MTA1mRNA表达量显著高于无淋巴结转移标本,差异有高度显著性(P＜0.01)。
4.45例食管癌组织中MTA1蛋白表达阳性率为55.6%(25/45),对应远癌正常组织中却未见MTA1蛋白表达阳性表达。18例伴有淋巴结转移食管癌组织中MTA1蛋白的阳性表达率为100%(18/18);而无淋巴结转移27例食管癌组织中MTA1蛋白的阳性率为25.9%(7/27),两组问差别有显著性(P＜0.05)。
第二部分:构建针对MTA1和Livin基因的siRNA载体及其沉默效果的分析
方法:
利用Takara、Ambion和Promega siRNA靶序列分析设计系统,扫描人MTA1 cDNA编码序列(NM-004689)和Livin cDNA编码序列(NM_139317),依据siRNA靶序列设计原则,经BLAST同源性分析,最终MTA1基因确定5个靶序列,Livin基因最终确定2个靶序列,同时随机组合出一条无关对照siRNA序列。分别合成8对编码短发卡RNA序列的DNA单链,两端分别加入BamHI和XhoⅠ的酶切残基。将合成的8对发卡DNA分别退火,分别克隆入siRNA载体(pRNAT-U6.2/Lenti),通过PCR筛选和DNA序列分析,得到重组子pRNAT-U6.2/Lenti-M286、pRNAT-U6.2/Lenti-M481、pRNAT-U6.2/Lenti-M533、pRNAT-U6.2/Lenti-M1018、pRNAT-U6.2/Lenti-M1332、pRNAT-U6.2/Lenti-L440、pRNAT-U6.2/Lenti-L652和pRNAT-U6.2/Lenti-Con。与辅助包装质粒共转染293FT细胞,进行病毒包装;收集、纯化得到重组慢病毒颗粒。分别感染食管癌EC9706细胞,G418筛选得到稳定感染细胞株。分别用荧光定量RT-PCR和Western blot检测MTA1或Livin基因表达沉默效果。
结果:
1.筛选得到5个针对MTA1基因的siRNA靶序列,286-304nt,481-499 nt,533-551 nt,1018-1036 nt,1332-1350 nt;2个针对Livin基因的siRNA靶序列,分别为440-458 nt和652-470 nt。
2.成功构建5个针对MTA1基因、2个针对Livin基因和1个无关序列慢病毒siRNA表达载体(pRNAT-U6.2/Lenti-M286、pRNAT-U6.2/Lenti-M481、pRNAT-U62/Lenti-M533、pRNAT-U6.2/Lenti-M1018、pRNAT-U62/Lenti-M1332、pRNAT-U62/Lenti-L440、pRNAT-U6.2/Lenti-L652和pRNAT-U6.2/Lenti-Con),包装纯化得到重组慢病毒颗粒。
3.经过G418筛选感染细胞,得到5株稳定感染沉默MTA1表达的EC9706细胞株(siM286、siM481、siM533、siM1018和siM1332),得到2株稳定感染沉默Livin表达的EC9706细胞株(siL440和siL652)。
4.感染靶向MTA1基因重组siRNA慢病毒的实验组食管癌EC9706细胞中MTA1 mRNA和蛋白表达都受到抑制,但抑制程度不同。其中pRNAT-U6.2/Lenti-M481包装产生的慢病毒,感染EC9706细胞的抑制效果最佳,MTA1mRNA和蛋白表达几乎完全抑制。
5.感染靶向Livin基因重组siRNA慢病毒的实验组食管癌EC9706细胞中LivinmRNA和蛋白表达都受到抑制。pRNAT-U6.2/Lenti-L440包装产生的慢病毒,感染EC9706细胞的抑制效果强于pRNAT-U6.2/Lenti-L652,Livin表达几乎完全抑制。
第三部分:RNAi干扰MTA1和Livin基因表达对食管癌EC9706细胞凋亡和侵袭迁移的影响
方法:
选用第二部分已构建出最有效沉默MTA1和Livin基因的siRNA载体,XhoⅠ和PmeⅠ双酶切pRNAT-U6.2/Lenti-Livin,回收XhoⅠ粘端和PmeⅠ平端线形化载体;AccⅡ和XhoⅠ酶切pRNAT-U6.2/Lenti-MTA1,回收475bp的MTA1siRNA单元:将MTA1 siRNA单元克隆入线形化pRNAT-U6.2/Lenti-Livin,经PCR筛选鉴定得到Livin和MTA1双基因沉默siRNA载体(pRNAT-U6.2/Lenti-Livin-MTA1)。与辅助包装质粒共转染293FT细胞,进行病毒包装;收集、纯化得到重组Livin和MTA1双基因沉默慢病毒颗粒。感染食管癌EC9706细胞,G418筛选得到稳定感染Livin和MTA1双基因沉默细胞株;荧光定量RT-PCR和Western-blot检测其沉默效果。对单一Livin基因沉默细胞株、单一MTA1基因沉默细胞株和Livin、MTA1双基因沉默细胞株,分别测定细胞生长曲线,Caspase-3活性,检测细胞凋亡和放射线敏感性的影响;测定细胞侵袭迁移能力。
结果:
1.构建出针对Livin和MTA1双基因沉默慢病毒siRNA载体pRNAT-U6.2/Lenti-Livin-MTA1,包装出Livin和MTA1双基因沉默慢病毒颗粒。
2.筛选得到稳定Livin和MTA1双基因沉默的食管癌细胞株。
3.Livin和MTA1双基因沉默慢病毒感染食管癌EC9706细胞,能够有效抑制细胞Livin和MTA1基因mRNA和蛋白的表达,与单一沉默Livin基因和MTA1基因的抑制效果相当。
4.单一Livin基因沉默和Livin、MTA1双基因沉默,可使食管癌EC9706细胞生长速度显著减缓,凋亡率、Caspase-3活性和放射线敏感性显著增加,差异均有显著性(P＜0.05)。
5.单一MTA1基因沉默和Livin、MTA1双基因沉默,可使食管癌EC9706细胞划痕愈合减缓和穿透胶能力降低。并且,Livin、MTA1双基因沉默细胞穿透胶能力降低的效果显著优于单一MTA1基因沉默细胞。
第四部分:Livin和MTA1双基因沉默EC9706细胞裸鼠移植瘤的实验研究
方法:
用第三部分建立的Livin和MTA1双基因沉默EC9706细胞株作为实验组,以第二部分建立的无关siRNA对照食管癌EC9706细胞株作为无关siRNA对照组,用未感染的食管癌EC9706细胞株作为空白对照组。培养上述3种细胞,分别接种5周龄的雌性BALB/c裸鼠。测量肿瘤大小绘制肿瘤生长曲线,28天后处死裸鼠,取肿瘤组织称重。荧光定量RT-PCR检测移植瘤组织中Livin、MTA1和PCNA mRNA的表达;免疫组化法检测移植瘤组织中Livin、MTA1蛋白的表达;流式细胞术检测移植瘤细胞的凋亡情况。
结果:
1.MTA1和Livin双基因沉默的食管癌EC9706细胞移植瘤,瘤体生长速度减慢。
2.MTA1和Livin双基因沉默的食管癌EC9706细胞移植瘤平均重量为228.1mg,两个对照组分别为1265.6mg和1341.8mg,组间比差异有显著性(P＜0.05)。
3.MTA1和Livin双基因沉默的食管癌EC9706细胞移植瘤细胞中PCNAmRNA表达量比两个对照组PCNA mRNA表达量显著降低,差异有显著性(P＜0.05)。
4.MTA1和Livin双基因沉默的食管癌EC9706细胞移植瘤细胞凋亡率为12.75%,而2个对照组细胞凋亡率分别为1.53%和1.68%,差异有显著性(P＜0.05)。
结论:
1.食管癌组织中存在MTA1和Livin基因呈现高表达;MTA1基因表达与食管癌淋巴结转移密切相关。MTA1蛋白表达水平可能成为判断食管癌淋巴结转移的重要指标。
2.成功构建出高效Livin和MTA1双基因沉默的慢病毒siRNA载体。
3.筛选得到稳定Livin和MTA1双基因沉默的食管癌EC9706细胞株:Livin、MTA1单一基因沉默的食管癌EC9706细胞株。
4.体外细胞实验证实,Livin和MTA1双基因沉默,可使食管癌EC9706细胞生长速度显著减缓,浸润和侵袭能力显著降低,肿瘤细胞凋亡率和放射线敏感性显著增加,并且效果优于单一基因沉默。
5.裸鼠体内实验证实,沉默食管癌EC9706细胞MTA1和Livin双基因的表达后,可以有效的抑制移植瘤的生长,并且增加了肿瘤细胞凋亡。
6.同时沉默Livin和MTA1基因的表达,有望成为食管癌靶向基因治疗的新方法。
Background and Aim
Livin was one member of IAP(inhibitor of apoptosis protein) family which can directly combine with Caspase(cysteine-containing aspartate-specific proteases), to play a role in antiapoptosis.The Livin expression in the placenta tissue and its elevated level in the embryonic developing tissues are found;while there is no detectable expression of Livin in the normal adult terminal differentiated tissue.The overexpression of Livin in most tumors can cause cells to resist apoptosis,which is closely correlated to tumorigenesis and tumor progression.With more and more studies Livin may become a novel molecule target for anti-apoptosis in tumor therapy.
Metastasis associated gene(MTA) is a gene family,including three kinds (MTA1,MTA2,MTA3)and six subtypes(MTA1,MTA1s,MTA-ZG29p,MTA2, MTA3,MTA3L).The MTA1 functions as a nucleosome remodeling and histone deacetylase(NuRD) component with nucleosome reconstitution and histone deacetylation enzyme activity.These characteristics can modulate gene transcription and replication procedures through ATP-dependent NuRD acetylation and deacetylation to affect the chromatin state,which may contribute to modulating the protein expression related to tumor metastasis and invasion via signal transduction and gene expression regulation.A lot of studies showed that the enhanced expression level of MTA1 had close relationship to cancer invasion and metastasis.The MTA1 gene may become an effective target for modulating malignant tumor invasion and metastasis.
With the development of gene therapy,RNA interference(RNAi) technique emerged.It is a phenomenon of post-transcriptional gene silencing with double stranded RNA,which causes effective silencing of homologous mRNA molecules, and also provides an effective way to inhibit the pathogenesis gene expression.It has been confirmed that some tumor biological behaviors could be reversed by interfering pathogenesis gene expression.However,multiple factors and genes may participate in tumorigenesis process,so that the expecting effect cannot be always carried out by interfering single molecule expression.Combination of Livin and MTA1 may be a significant factor for interfering tumorigenesis and malignant invasion,and some researchers have silenced them by RNAi,which exhibited some inhibiting effects on tumor.The aims of this study:①To detect Livin and MTA1 expression in the esophageal tumor tissue and to reveal the two molecules' relationship to tumor metastasis and invasion;②To construct silenced siRNA Lentivirus vectors targeting Livin and MTA1 and to construct the esophageal tumor cell models with silenced Livin and MTA1 genes.③To examine the cell growth, apoptosis,metastasis and invasion of the esophageal tumor cells after silencing Livin and MTA1 genes,and to provide some theoretical and experimental data to support targeting therapy of esophageal carcinoma.
PartⅠ:Analysis of the Livin and MTA1 Expressions in the Esophageal Carcinoma
Methods:
45 samples of esophageal tumor tissues were collected,the tumor focus and the normal mucous membrane tissues(near distal end 5cm) were obtained from each sample.The mRNA expression level of Livinα,Livinβand MTA1were detected by real-time RT-PCR,the protein expressions of Livin and MTA1 were assayed by immunohistochemistry.
Results:
1.The mRNA expression level of Livinαand Livinβdetected by RT-PCR in 45 samples of the esophageal tumor tissue was higher than that of the corresponding normal tissue,5 cm far from the tumors(P＜0.01);the expression level average of Livinβwas higher than that of Livinα,but there was no significant difference between them(P＞0.05).
2.The positive rate of Livin protein expression examined by immunohistochemistry was 95.6%(43/45) in 45 samples of the esophageal tumor tissue,while the positive rate of Livin protein expression was only 6.7%(3/45 ) in the corresponding normal tissue.There was significant difference between them(P＜0.01).
3.The mRNA expression level of MTA1 detected by RT-PCR in 45 samples of the esophageal tumor tissue was higher than that of the corresponding normal tissue (P＜0.01);the mRNA expression level of MTA1 in the samples with lymph node metastasis was remarkably higher than that of the samples without lymph node metastasis.There was significant difference between them(P＜0.01).
4.The positive rate of MTA1 protein expression examined by immuno -histochemistry was 55.6%(25/45) in 45 samples of the esophageal tumor tissue, but MTA1 protein expression was not found in the corresponding normal tissue. The positive rate of MTA1 protein expression was 100%(18/18) in 18 samples of the esophageal tumor tissue with lymph node metastasis,but it was 25.9% (7/27) in 27 samples without lymph node metastasis.There was significant difference between them(P＜0.05).
PartⅡ:Construction of siRNA Vectors Targeting Respective Livin and MTA1 Genes and Detection of their Silencing Effects
Methods:
According to the principle of designing siRNA sequence,the Takara,Ambion and Promega siRNA sequence for designing and analyzing software system was used to scan human MTA1 cDNA sequence(NM_004689) and Livin cDNA sequence(NM_139317).Five siRNA sequences targeting MTA1 and two siRNA sequences targeting Livin were analyzed by BLAST homology and a contrast sequence(Con) was randomly assorted.Eight DNA oligonucleotides of short hairpin RNA sequence were synthesized and annealed into double strands respectively with BamHI and XhoI restricted incision enzyme sites;eight hairpin DNAs were cloned into siRNA expression vector(pRNAT-U6.2/Lenti) that had been cut by BamHI and XhoI restriction enzymes;the siRNA expression vector (pRNAT-U6.2/Lenti-M286,pRNAT-U6.2-/Lenti-M481,pRNAT-U6.2/Lenti-M533, pRNAT-U6.2/Lenti-M1018,pRNAT-U6.2/Lenti-M1332,pRNAT-U6.2/Lenti-L440, pRNAT-U6.2/Lenti-L652 and pRNAT-U6.2/Lenti-Con) were obtained by PCR and DNA sequencing,which were transfected into 293FT cells with aiding packaging plasmid.The Lentivirus particles were obtained by packaging,collecting and purifying;The esophageal carcinoma cell line EC9706 were infected respectively by these Lentivirus particles,the infected stable cell lines were established by G418 screening;The silencing effect was detected by real time RT- PCR and Western blotting.
Results:
1.Five siRNA sequences targeting MTA1 were obtained(286-304nt,481-499nt, 533-551nt,1018-1036nt,1332-1350nt);Two siRNA sequences targeting Livin were obtained(440-458nt and 652-670nt).
2.Five Lentivirus siRNA expression vectors targeting MTA1(pRNAT-U6.2/Lenti -M286,pRNAT-U6.2/Lenti-M481,pRNAT-U6.2/Lenti-M533,pRNAT-U6.2/ Lenti-M1018,pRNAT-U6.2/Lenti-M1332),two Lentivirus siRNA vectors targeting Livin(pRNAT-U6.2/Lenti-L440 and pRNAT-U6.2/Lenti-L652) and a contrast Lentivirus siRNA expression vector(pRNAT-U6.2/Lenti-Con) were successfully constructed,and the recombinated Lentivirus particles were obtained by packaging and purifying.
3.Five infected stable EC9706 cell lines silencing MTA1(siM286,siM481,siM533, siM1018 and siM1332 ) and two infected stable EC9706 cell lines silencing Livin (siL440 and siL652 ) were establisked via G418 screening.
4.The mRNA and protein expressions of MTA1 in cells of each group transfected by the Lentivirus vectors targeting MTA1 were obviously inhibited,but their silencing effects were different;among them,the silencing effect of pRNAT-U6.2/Lenti-M481 was the best,they were about completely depressed.
5.The mRNA and protein expression of Livin in cells of each group transfected by the Lentivirus vectors targeting Livin were markedly inhibited,and the silenced effect of pRNAT-U6.2/Lenti-L440 was better than that of pRNAT-U6.2/Lenti -L652,they were about completely depressed.
PartⅢ:Effects on Apoptosis and Migration of Esophageal EC9706 Cancer Cells Silenced by Reconstructed Single siRNA Vector Targeting both Livin and MTA1 Genes
Methods:
Based on the siRNA vectors constructed and screened in the second part,the putative most effective siRNA vector,recombinant single siRNA vector targeting both Livin and MTA1 genes was constructed.The pRNAT-U6.2/Lenti-Livin was cut by XhoI and PmeI,then the lineafizated vector was reclaimed;pRNAT-U6.2/Lenti -MTA1 was cut by AccII and XhoI;MTA1 siRNA unit with 475bp was reclaimed and cloned into the lineafizated pRNAT-U6.2/Lenti-Livin.The pRNAT-U6.2-Livin-MTA1 was identified and obtained by PCR,which was transfected into 293FT cells with aiding packaging plasmid.The Lentivirus particles silencing both Livin and MTA1 were packaged,collected and purified.The EC9706 cells were transfected by these Lentivirus particles.The infected stable cell lines were established via G418 screening respectively.The silencing effect detected by real time RT-PCR and Western blotting;the apoptosis effect detected by flow cytometry,the growth curve and sensitivity to radioactive ray detected by MTT assay,the migration ability detected by Matrigel Invasion Assay and the Caspase-3 activity detected by related kit were examined in respective established stable cell lines,targeting single Livin,targeting single MTA1 and targeting double Livin and MTA1.
Results:
1.The Lentivirus vector targeting both Livin and MTA1(pRNAT-U6.2/Lenti -Livin-MTA1) was successfully constructed;the Lentivirus particles silencing Livin and MTA1 were packaged.
2.The stable esophageal carcinoma cell line silencing both Livin and MTA1 genes was obtained by screening.
3.Both mRNA and protein expressions of Livin and MTA1 genes were inhibited in the EC9706 cells transfected by the Lentivirus particles silencing both Livin and MTA1 genes,and the silencing effect was similar to that of silencing Livin or MTA1 alone.
4.The cell growth velocity was reduced,while the apoptosis rate,the Caspase-3 activity and the sensitivity to radioactive ray obviously was increased in the EC9706 cell lines,containing silencing single Livin and silencing both Livin and MTA1.There was significant difference between the experimental group and the control group(P＜0.05).
5.The Matrigel invasion ability repressed in the EC9706 cell lines,silencing single MTA1 and silencing both Livin and MTA1.Besides,the repressing effect of Matrigel invasion ability in the cells silencing both Livin and MTA1 was better than that of the cells silencing MTA1 alone.
PartⅣ:The Nude Mouse Transplanted Tumor with EC9706 Cells Silencing both MTA1 and Livin Genes
Methods:
The EC9706 cell line silencing both MTA1 and Livin,the irrelevant siRNA EC9706 cell line and the untransfected EC9706 cell line were considered as the experimental group,the irrelevant siRNA control group and the untransfected control group respectively.Three above-mentioned cell lines were cultivated and inoculated into the female BALB/c nude mice aged 5 weeks old.The tumor growth curve was determined by measuring the tumor size;the nude mice were killed 28 days later and the tumors were weighted.The mRNA and protein expressions of Livin,MTA1 and PCNA were detected by real time PCR and immunohistochemistry;the apoptosis was detected by flow cytometry.
Results:
1.The tumor growth speed was retarded in the transplanted tumor with EC9706 cell line silencing both MTA1 and Livin.
2.The average weight of the transplanted tumor with EC9706 cell line silencing both MTA1 and Livin was 228.1 mg,the average weights were 1265.6 mg and 1341.8 mg in the other two control groups respectively.There was significant difference between them(P＜0.05).
3.The mRNA expression level of PCNA in the transplanted tumor with EC9706 cell line silencing both MTA1 and Livin was lower than that of the two control groups.There was significant difference between them(P＜0.05).
4.The cell apoptosis rate was 12.75%in the transplanted tumor with EC9706 cell line silencing both MTA1 and Livin;the cell apoptosis rates were 1.53%and 1.68%respectively in the other two control groups.There was significant difference between them(P＜0.05).
Conclusions
1.There are elevated expression level of Livin gene and MTA1 gene in the esophageal carcinoma tissue;and the MTA1 expression level is closely related to lymph node metastasis of the esophageal carcinoma.The protein expression level of MTA1 may be an important marker for evaluating lymph node metastasis of the esophageal carcinoma.
2.The effective Lentivirus vector silencing both Livin and MTA1 genes was constructed successfully.
3.The stable EC9706 cell lines silencing both Livin and MTA1 genes,silencing single Livin gene and silencing single MTA1 gene were established respectively.
4.The experiment in vitro indicates that silencing both Livin and MTA1 genes can reduce the cancer cell growth and invasion capacity;while apoptosis and sensitivity to radioactive ray markedly enhance,its effect is better than that of silencing Livin or MTA1 gene alone.
5.The experiment on nude mice exhibits that silencing both Livin and MTA1 genes can inhibit growth of transplanted tumor and facilitate the tumor apoptosis.
6.To silence Livin and MTA1 may provide a novel strategy for target gene therapy of the esophageal carcinoma.
引文
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