| Background and AimLivin 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:â‘ To detect Livin and MTA1 expression in the esophageal tumor tissue and to reveal the two molecules' relationship to tumor metastasis and invasion;â‘¡To construct silenced siRNA Lentivirus vectors targeting Livin and MTA1 and to construct the esophageal tumor cell models with silenced Livin and MTA1 genes.â‘¢To 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.Partâ… :Analysis of the Livin and MTA1 Expressions in the Esophageal CarcinomaMethods: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).Partâ…¡:Construction of siRNA Vectors Targeting Respective Livin and MTA1 Genes and Detection of their Silencing EffectsMethods: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(NM004689) and Livin cDNA sequence(NM139317).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.Partâ…¢:Effects on Apoptosis and Migration of Esophageal EC9706 Cancer Cells Silenced by Reconstructed Single siRNA Vector Targeting both Livin and MTA1 GenesMethods: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.Partâ…£:The Nude Mouse Transplanted Tumor with EC9706 Cells Silencing both MTA1 and Livin GenesMethods: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).Conclusions1.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.
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