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Objective To explore tumor inhibitory effect of the lentiviral vectors mediated small interfering RNA(siRNA)targe‐ting survivin gene on human lung adenocarcinoma xenografts in nude mice in vivo .Methods The lentiviral vector expressing sur‐vivin‐siRNA was prepared .Then established human lung adenocarcinoma‐bearing nude mice model ,and divided nude mice into three groups ,including the blank control group(PC group) ,blank vector group(NC group) and experiment group(RNAi group) ,treated with physiological saline ,blank viral vector and siRNA respectively .The lentiviral vectors expressing survivin‐siRNA were locally injected in tumor issues of nude mice in the RNAi group .Then observed the curve about changes of tumor volume in different time . The expression levels of protein and mRNA were detected by using Western blot and reverse transcription polymerase chain reac‐tion(RT‐PCR) .The histogram of tumor cell cycle were examined by using flow cytometry .Results The rate of tumor inhibition of lentiviral vectors mediated survivin‐siRNA on lung adenocarcinoma in nude mice was 46 .07% .There were significant differences in tumor weight between siRNA group and NC group ,PC group(P0 .05) .Compared with NC group and PC group ,the expression levels of survivin mRNA and protein were decreased ,and rates of inhibition were 72 .00% and 53 .00% respectively .The percentage of G1 phase cells was in‐creased ,whereas the percentages of S phase cells was decreased .Conclusion The lentiviral vectors mediated siRNA could effective‐ly inhibit survivin gene expression on human lung adenocarcinoma xenografts in nude mice and markedly induce the apoptosis of tumor .
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Objective To observe the effect of rhTRAIL on survivin gene expression of human lung adeno-carcinoma A549 xenografted tumor in nude mice,and investigate the possible inhibitory mechanism of rhTRAIL on the implanted-tumor growth.Methods The solid tumor model was formed in nude mice with human lung adeno-carcinoma cell line.A549.24 mice were randomly divided into the four groups,rhTRAIL single treated group (1 μg/mL),rhTRAIL combined with cisplatin (DDP) treated group,cisplatin treated (1.5mg/kg) and 0.9％ sodium chloride injection(NS) control group.The rhTRAIL and DDP were injected once every other day by intraperitoneal injection to mice in the treated groups,lasting eight times,the same volume of saline solution was injected to the control group.After these,mice were killed and dissected completely.The expression level of survivin mRNA and protein in the tumor tissues was detected by real-time polymerase chain reaction (RT-PCR) and immunohistochemistry,respectively.And the expression of survivin gene in serum of each group was tested by ELISA.Results The expression levels of survivin mRNA in implanted-tumor tissues in rhTRAIL,rhTRAIL combined with DDP,DDP and NS group were (48.7 ± 2.5) ％,(53.1 ± 4.6) ％,(99.1 ± 5.3) ％ and (95.6 ± 3.7) ％,respectively.While the protein expressions of survivin gene in those groups were (0.319 ± 0.025),(0.483 ± 0.058),(0.635 ± 0.041) and (0.619 ± 0.017),respectively.Moreover,the serum levels of survivin were (71.9 ± 7.05),(80.26 ± 10.80),(112.75 ± 15.41) and (105.03 ± 20.37),respectively.The data showed that the expression levels of rhTRAIL and rhTRAIL combined with DDP group were lower than that of DDP-treated group or the NS control group (P ＜ 0.0 5).Compared with the rhTRAIL combined with DDP group,the survivin gene expression level of rhTRAIL-single treated group decreased a little lower,but the difference was not significant (P ＞ 0.05).Conversely,the survivin gene level was increased to some degree compared with the NS control group,and uniformly there was no significant difference (P ＞ 0.05).Conclusion rhTRAIL can downregulate the expression level of survivin gene of human lung adeno-carcinoma A549 xenografted tumor in nude mice.It may be one of the possible inhibitory mechanisms of rhTRAIL on the implanted-tumor growth that rhTRAIL can downregulate survivin gene expression and promote tumor cell apoptosis.
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Background and purpose: The effects of tumor after radiotherapy and chemotherapy with tumor cell apoptosis have positive correlation. Survivin is an apoptosis suppressor gene, which is overexpressed in lung cancer. The reduction of its expression can increase lung cancer cell apoptosis. RNA interference can speciifcally and effectively blocked the expression of this gene. After accepting a dose of radiation, tumor tissues can raise the rate of gene transduction. The purpose of this study was to investigate the effects of the expression of Survivin gene suppressed by intratumor injection siRNA and radiation in lung cancer. Methods:Mouse subcutaneous transplanted with tumors were randomly divided into four groups:untreated group (group A), group with intratumor injection siRNA (group B), group with radiation (group C), group 4 Gy pre-irradiation combination with intratumor injection siRNA (group D). Two days later, mouse with different treatments were executed by cervical dislocation and stripped the subcutaneous tumors. mRNA and protein levels of Survivin gene were detected by quantitative real-time PCR (qRT-PCR) and Western blot; Cell cycle and cell apoptosis were assayed by lfow cytometry (FCM). Results:The expression of Survivin gene in mRNA and protein levels in group B, C, and D were signiifcantly reduced compared with group A. The differences of Survivin expression in mRNA level between group A and group D were statistically signiifcant. Compared with the other 3 groups, the expression of Survivin gene in group D was signiifcantly reduced, the differences were statistically signiifcant (P=0.036);The proportion of cells in S-phase of group D was (2.70±0.34)%, compared with group B [(8.93±0.75)%] and group C [(6.71±0.51)%], that was significantly reduced. The differences of the proportion of S-phase cells in group A and group D were statistically signiifcant (P=0.034);The rate of cell apoptosis in group D was (25.67 ± 0.65)%, which was signiifcantly increased compared with the rate of cell apoptosis in the other 3 groups, the differences were statistically signiifcant (F=78.82, P<0.05). Conclusion:Pre-irradiation can enhance the transduction rate of siRNA, reduce the expression of Survivin gene in lung cancer, promote cell apoptosis, and increase the sensitivity of the radiotherapy.
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Objective To investigate the biological response of survivin siRNA in A549 human lung cancer cells and Hela S3 human cervical cancer cells as well as K562 human erythroleukemia cells,in order to screen the working sequences of survivin siRNA.Methods Three sequences of survivin-targeted siRNA were designed and synthetized,and human cancer lines of A549,Hela S3,K562 were transfected with Hiperfect liposome entrapped survivin siRNA,respectively.The expression of survivin mRNA was detected by means of real-time polymerase chain reaction (RT-PCR) with SYBR Green Ⅰ.Cell proliferation was detected by way of WST-8 cell count kit at 48 hours and 72 hours after transfection.Results The expression of survivin mRNA in all 3 cancer cells studied was significantly inhibited by all siRNA at 48 hours and 72 hours after transfection,gene expression inhibition ratio were 57.47％-88.53％ after transfection 48 hours and were 69.94％-95.03％ after transfection 72 hours.Cell proliferation was also significantly inhibited 48 hours and 72 hours after transfection,cell multiplication inhibition ratio were 27.88％-47.36％ after transfeotion 48 hours and were 42.59％-57.29％ after transfeciton 72 hours.Sequence 1 had the most inhibition efficacy on survivin gene expression and proli-feration in tumor cells.Inhibition rate of the three tumor cell gene expression of survivin at 48 hours are above 75％,72 h all over 90％,48 hours of cell proliferation inhibition rate are above 40％,72 hours of more than 50％.Conclusions The chemo-synthesized siRNAs can significantly down-regulate survivin mRNA expression in cancer cell studies.Survivin siRNA is capable of inhibiting the proliferation of tumor cell in vitro and it might be a new strategy for tumor-targeted therapy.Sequence 1 is the most efficacious working survivin siR-NA in the study.
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Survivin is an apoptosis inhibitor and plays an important role in the development and progression of cancer.It is undetectable in normal adult tissues,but has been showed to be overexpressed in various cancers and has been regarded as a marker of malignancy.And the promoter polymorphism of Survivin gene is closely related to risk of cancer and gene susceptibility,this might provide clues for further elucidation of the correlation of risk of cancer and genetic polymorphism,and for diagnosis,prevention and screening of cancer gene.
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Objective: To explore the association of survivin gene with hepatocellular carcinoma(HCC) in Han nationality in east China. Methods: A case-control study was designed, which included 176 HCC cases and 196 healthy controls, all from Haimen city of Jiangsu province. The rs1042489 and rs9904341 loci of survivin gene were genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique, and the association of the genotypes and haplotypes of the two loci with HCC was analyzed under different genetic models. Results: Univariate analysis showed no significant difference in the genotypes or allele distribution between HCC case group and the control group under different genetic models in either rs9904341 or rs1042489 loci (P>0.05). Linkage disequilibrium (LD) analysis showed that the two loci were in LD (χ2 = 4.777, P = 0.03), with the D' value being 0.188 and 0.183 in the case and control group, respectively. The recessive genetic model was found to be the optimal model by multivariate Logistic regression, according to the lowest value of Akaike's information criteria (AIC). With no rs9904341C-rs1042489T (C-T) haplotype as reference, it was found that the haplotype of C-T from the two loci was associated with a lower risk for HCC under the recessive genetic model, with the factors such as alcohol drinking and HBV infection controlled (OR = 0.48, P = 0.04), and no other haplotypes were found associated with HCC. Multivariate analysis showed that HBsAg+ and hepatitis B were the risk factors for HCC, but without haplotype-environment interaction. Conclusion: No association has been found for rs9904341 or rs1042489 in survivin gene with HCC in Han nationality in east China, and rs9904341C-rs1042489T might be a protective haplotype for HCC.
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[Objective] To investigate the association between -31C/G polymorphism in the promoter of survivin gene and the susceptibility to sporadic colorectal cancer in southern Chinese population. [Methods] Survivin-31C/G genotypes were determined by polymerase chain reaction-restriction fragment length polymorpbism (PCR-RFLP) in 711 healthy controls and 702 CRC cases. [Results] The number of CRC patients carrying with CC genotype was much higher than those of controls (36.5 % vs. 26.12%, X~2=17.89, P<0.001). Compared with CC genotypes, CG, GG genotypes and G allele carriers had a significantly decreased risk of CRC, with the decrease being 0.61-fold (95% CI=0.46-0.81, P<0.001), 0.52-fold (95% CI=0.38-0.71, P<0.001) and 0.58-fold (95% CI=0.45-0.74, P<0.001), respectively. [Conclusion] Survivin gene -31C/G polymorphism is associated with sporadic CRC risk, the G variant genotypo is the independent protective factors against sporadic CRC in soutbem Chinese population.
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Objective To investigate the expression and clinical significance of survivin and PTEN in bladder transitional cell carcinoma(TCC).Methods The expression of survivin and PTEN was studied by S-P immunohistochemisty in 10 cases of normal urinary bladder tissues and 62 cases of the bladder TCC. Results All the normal urinary bladder tissues showed positive staining pattern of PTEN protein, negative staining pattern of survivin protein, positive rate of PTEN in 62 cases of the bladder TCC is 46.8%,the difference of positive rate between grade Ⅰ and grade Ⅲ was significant(P＜0.05),the difference of positive rate between Tis～T_1 and T_2～T_4 showed a trend of decreasing, but there was no statistically significant difference(P＞0.05);positive rate of survivin in 62 cases of the bladder TCC is 56.5%,the difference of positive rate between grade Ⅰ and grade Ⅲ was significant(P＜0.05),the difference of positive rate between Tis～T_1 and T_2～T_4 was significant too(P＜0.05).The expression of PTEN protein and survivin protein was reversely correlated(P＜0.05).Conclusion The abnormal expression of PTEN protein and survivin protein play an important role in the occurrence and progress of the bladder TCC.It can be regarded as a useful diagnostic marker in the bladder TCC.
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Objective To construct an eukaryotic vector with expression of human survivin gene with green fluorescent protein which is named pIRES2-EGFP/survivin and transfected into K562 cell line.Methods Using pDNR/Survivin plasmid as a template, the full length of survivin cDNA was amplified by PCR and subsequently cloned into T-A vector and then subcloned into pIRES2-EGFP vector. After identified by digestion of restrictive endonucleases, pIRES2-EGFP/survivin was further confirmed by sequencing. Then it was transfected into K562 cells with superfect reagents. The mRNA was isolated and survivin gene was detected by Western blotting. Results The exact sequences of pIRES2-EGFP/survivin vector were confirmed by digestion of restrictive endonucleases and sequencing. After transfection, the expressions of green fluorescent protein were present. The mRNA expression of survivin has been detected in transfected cells by RT-PCR. Conclusion The vector pIRES2-EGFP/survivin has been constructed and could express survivin gone in K562 cells successfully.
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Objective To explore the mechanisms of apoptosis induced by arsenic trioxide and amifostine in human acute promyelocytic leukemia cell lines HL-60 in vitro.Methods HL-60 cells were treated with different concentrations of arsenic trioxide alone and combined with amifostine.The inhibitory ratio of the ceils were measured by MTT assay.and the expression of Survivin Was detected by semiquantitate RT-PCR.Results Proliferation of HL-60 cells exposed to arsenic trioxide dwpped down with increasing dose of the dmg and this effect Was significantly hisher when arsenic trioxide Was used in combination with amifostine.Furthermore.there was a more significant decrease in Survivin expression in HL-60 cells treated with arsenic trioxide in combination with amifostine as compared to the cells treated only with arsenic trioxide.Conclusion Arsenic trioxide induced HL-60 cells to undergo apoptosis by downregulating the expression of Survivin. Amifostine enhanced the sensitivity of HL-60 cells to arsenic trioxide by downregulating the expression of Survivin,thus promoting apoptosis effect.
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Objective:To use polyamidoamine(PAMAM)dendrimer as gene delivery system for survivin gene anti- sense oligodeoxynucleotide(asODN)transfection for inhibition of HepG2 cancer cell growth.Methods:The first to the fifth generation of PAMAM and asODN were used to prepare a complex:PAMAM-asODN.The morphology of PAMAM- asODN was observed using agrose electrophoresis and atomic force microscope(AFM).PAMAM-asODN was then used to transfect HepG2 cells and cells transfected with asODN served as control.The transfection efficacy of PAMAM-asODN into HepG2 cells was observed under confocol microscope,the surviving mRNA expression was analyzed by RT-PCR,and the inhibition of HepG2 cell growth was determined by MTT assay.Results:Agrose electrophoresis showed strong complexing action between PAMAM and asODN and they formed a complex with a diameter of 25 nm.Confocol microscope showed the transfection efficacy of PAMAM-asODN was higher than that of asODN.RT-PCR showed a decreased expression of sur- vivin mRNA in PAMAM-asODN transfected cells.MTF results demonstrated that the growth of HepG2 cell was obviously inhibited after transfection of PAMAM-asODN and the inhibition rate increased with culture time,concentration of com- plex,the generation of PAMAM.PAMAM-asODN at 6.0?mol/L G4.0 resulted in a 55% inhibition of HepG2 cells 96 h after culture.Conclusion:PAMAM dendrimers can efficiently mediate the entry of survivin asODN into HepG2 cells,re- sulting in inhibition of HepG2 cells.PAMAM might be a promising gene carrier for potential molecular therapy of cancer.
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Objective To study the activity of the survivin gene promoter in several tumor cell lines and evaluate the possible application of this promoter in tumor gene therapy. Methods ①The expressions of survivin gene in A549,MDA-MB231 and HepG2 cell lines were detected by RT-PCR and Western blotting.②Tumor cells(A549,MDA-MB231,HepG2) were transiently transfected by reporter plasmids containing different length of survivin promoter using lipofectamine.And 48 h later,the level of reporter gene expression was analyzed.Results There were different levels of survivin expression in A549,MDA-MB231 and HepG2 cell lines.Transient transfection assay approved that pLuc-surP-987,pLuc-surP-596,pLuc-surP-269 and pLuc-surP-158 showed high activity and 269 bp survivin promoter demonstrated the highest activity. Conclusion In transcriptional level,survivin promoter can activate the reporter gene in several tumor cell lines.It is a potential candidate promoter in tumor gene therapy.
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Objective To investigate the expressions of platelet derived growth factor (PDGF) and survivin in hepatocellular carcinoma (HCC) with portal vein tumor thrombosis (PVTT) and to evaluate the relationships among the expressions of PDGF, survivin and the proliferation of cancer cells. Methods The expression of PDGF mRNA in 16 cases in HCC with PVTT group was observed by in situ hybridization and the results were compared with that in HCC tissue group. The expressions of PDGF and survivin protein in 36 cases in HCC with PVTT group were detected with immunohistochemistry and it was found that there was a correlation between them. Flow cytometry (FCM) was used to measure the proliferation of cancer cells and it was also used to analyze the relations among PDGF, survivin and the proliferation of cancer cells. Results The expression level of PDGF mRNA in HCC with PVTT group was significantly higher than that in HCC tissue group (P
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Objective To construct the bicistronic retroviral vectors containing GFP gene and human survivin gene(wildtype and T34A mutant). Methods Survivin fragments were amplified from eukaryotic expression vectors by PCR, then cloned into pMIG retroviral vectors. The constructed recombinant was identified by double digestion with restriction enzymes. The constructed retroviral vectors was transfected into Phoenix E package cells under mediation of liposome. The expression of mRNA was examined by RT-PCR and the transcription of gene was determined by FACS analysis of GFP. Results The target fragments were successfully bound to pMIG vector. After the vectors were transfected into NIH3T3 cells, the expression of mRNA was confirmed by RT-PCR and the expression of GFP was confirmed by FACS analysis. Conclusion Human survivin gene wildtype and T34A mutant retroviral vectors have been constructed successfully, which can provide strong molecular tools for further studies and a novel approach for selective cancer gene therapy.
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Objective: To construct the eukaryotic expression vector pSurp-EGFP regulated by the survivin gene promoter and to detect the specific expression of the promoter in human laryngeal cancer Hep-2 cells by green fluorescent protein assay.Methods: Thesurvivin gene promoter was generated by polymerase chain reaction(PCR) and the CMV promoter of the pShuttle vector replaced by the survivin gene promoter to generate the plasmid pSurp.The three plasmids pShuttle,pSurp and pEGFP-C1 were respectively double-enzyme digested so as to produce the plasmids pCMV-EGFP and pSurp-EGFP carrying the CMV or survivin promoter.The purified pCMV-EGFP and pSurp-EGFP were transfected into Hep-2 cell and vascular endothelial cell ECV304 using liposome transfection reagent and the expressions of EGFP detected by the fluorescent microscope.Results: Thesurvivin gene promoter was successfully cloned by PCR,and thesurvivin gene promoter-regulated pSurp-EGFP was constructed.Green fluorescence was observed in Hep-2 cells but not in ECV304. Conclusion: The high specific activity of the survivin gene promoter in Hep-2 cells that we successfully constructed attributes to the studies of tumor specific gene therapy.
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AIM:To construct eukaryotic expression vector expressing double shRNA sections targeting Survivin gene.METHODS: Eukaryotic expression vector expressing double shRNA sections targeting Survivin gene were designed and chemically synthesized.They were directionally inserted into plasmid pGenesil-1 with respectively U6 promoter and termination code,the common green fluorescence protein(EGFP) gene and Neo gene. In this way,the vector of pGenesil-1 shRNA containing 2 sections of Survivin shRNA were constructed and they were transfected into the pancreatic cancer cell Bx-PC3.Transfection was detected by fluorescence microscope.The inhibition expression of Survivin mRNA was measured by RT-PCR.RESULTS: HE1 and HE2 plasmids were identified by the biocatalyst cut which confirmed the exactitude and were analyzed by the sequence analysis which verified the perfect clone plasmid inserted by them.CONCLUSION: A eukaryotic expression vector of double short hairpin RNA for Survivin gene is successfully constructed.The pancreatic cancer cells Bx-PC3 succeed to be transfected and expression of Survivin mRNA is inhibited obviously.
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Objective To study the influence of siRNA targeting survivin on chemotherapy sensitivity of HCC cells.Methods siRNA eukaryotic expression vector was generated.After the vector stablely transfected into HepG2 cells,the effects of chemotherapy drugs on HCC cells were observed.Results The recombinant plasmid Psilence(+)-survivin was successfully constructed.Survivin mRNA expression inhibition ratio reached 73% detected by RT-PCR.MTT methods detected that siRNA treated HCC cells were affected by MCC,the survival rate of HepG2,HepG2/Silence(-) cells was inhibited only at 48 h(0.505?0.015) compared to control groups untreated with MCC(0.824?0.322)(P
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Purpose: To investigate the expression of Survivin gene and its relationship with expression of Caspase-3, B-cell lymphoma-2( Bcl-2) in human non-small-cell lung carcinoma( NSCLC) . Methods: Expression of the Survivin mRNA was evaluated by in situ hybridization (ISH) in 13 normal bronchial epithelium, 11 dysplasia, 54 NSCLC and 12 lymph node metastasis. Immunohistochemical assay was used to detect the expression of Caspase-3 and Bcl-2 proteins. Results: Expression of Survivin was detected in a significantly greater proportion in NSCLC (74. 07%) and lymph node metastasis (91. 67%) than normal bronchial epithelium (7. 69%) and dysplasia ( 27. 27%) (P
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Objective:To study the expression of survivin gene in cervical carcinoma and its relation to the pathogenesis of cervical carcinoma.Methods:Using SP immunohistochemical technique,we examined 74 cervical tissues of clinicopathological specimens,including 10 normal cervical tissues,5 cervical intraepithelial meoplasia(CIN)lesions,59 invasive squamous cell carcinomas.Rusults:Expression of survivin were not observed in normal cervical tissues. With the lesions progressing ,the expression rate of survivin protein had a increasing tendency.Expression of survivin was related to the tumor grade and clinical stage.Conclusion:The results suggested that survivin expression is the essential event in carcinogenesis and development of cervical carcinoma.Survivin expression in cervical carcinoma is a useful prognostic marker.
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Objective To explore the efficacy of calmodulin antagonist berbamine(BBM) in down-regulating survivin mRNA. Methods Human breast cancer cell line MCF7 and its adriamycin-resistant counterpart MCF7/ADR were used in the study. The cells were cultured with different concentration of BBM for 72 hours. The mRNA expression level of survivin gene in both MCF and MCF7/ADR cells was detected by semi-quantitative RT-PCR. Results After treating MCF7 and MCF7/ADR cells by 20?mol/L BBM, the mRNA expression level of survivin gene decreased from 0.43?0.02 to 0.21?0.04 in MCF7 cells, and from 0.57?0.05 to 0.45?0.04 in MCF/ADR cells(P