腺病毒介导的RNA干扰ERCC1基因表达逆转卵巢癌顺铂耐药的研究
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摘要
卵巢癌是严重威胁妇女生命的恶性肿瘤,长期以来,尽管各种诊断与治疗方法不断完善和提高,使患者预后有所改善,但五年生存率仍较低,徘徊在25%～30%左右。由于多数卵巢恶性肿瘤对化疗敏感,特别是对以顺铂为基础的联合化疗敏感,使顺铂在卵巢癌的治疗中占有重要地位。但化疗耐药性的产生严重阻碍了化疗药物的抗肿瘤效应,其中以ERCCl基因高表达导致的顺铂耐药尤为重要。近年来发现的RNA干扰(RNA interfering,RNAi)是一种有效的基因沉默技术,能特异性地沉默靶基因的表达,在基因功能研究和抗肿瘤治疗方面得到了广泛应用。本实验利用RNA干扰技术,以腺病毒为载体,将针对ERCCl基因的外源性发夹环小干扰RNA片断导入腺病毒载体,通过基因重组技术构建稳定表达ERCCI-shRNA的重组体腺病毒,联合顺铂用药,体外实验观察腺病毒介导的RNA干扰ERCCl基因表达对卵巢癌顺铂耐药的逆转作用,为卵巢癌基因治疗逆转化疗耐药提供新的治疗途径。
第一部分ERCCl基因在卵巢癌细胞系中的表达及对卵巢癌细胞耐药的影响
目的:观察人卵巢癌细胞系中ERCCl基因的表达与卵巢癌顺铂耐药的关系。方法:应用RT-PCR方法检测ERCCl基因mRNA在卵巢癌细胞系COCl、COCl/DDP、A2780及A2780/TS中的表达,观察不同浓度顺铂作用24小时后基因表达的变化。结果:1、四种卵巢癌细胞系COCl、COCI/DDP、A2780及A2780/TS中均有ERCCI基因的表达,耐药亚系COCl/DDP和亲本COCl相比,前者ERCCl基因的表达量明显高于后者,统计学检验有显著差异(p<0.05);耐药亚系A2780/TS和亲本A2780相比,ERCCl基因的表达前者比后者略有升高,无统计学意义。2、不
Ovarian cancer is a malignant cancer which seriously threatens women health. Although prognosis has been improved with development of various diagnostic and therapeutic methods, 5 year of survival rate still remains 25%-30%. Since the chemotherapy is sensitive to most of malignant ovarian cancer, especially cisplatin-based chemotherapy. It has made cisplatin play a key role in treatment of ovarian cancer. However, the resistance of chemotherapy tremendously obstructs anticancer effect of chemotherapy drug, essentially, high expression of ERCC1 gene makes cisplatin resistant. Recently found RNA interfering (RNAi) is an effective technique of gene silence, it is specifically able to make target gene expression in silence, and it has been extensively used in investigation of gene function and anticancer drugs.Adenovirus-mediated RNA interfering technique which aims at inserting exogenous small hairpin interfering RNA will be used in our study. Meanwhile, the recombinant adenovirus which stable expresses ERCC1-shRNA will be constructed by gene recombinant technology, combination with cisplatin therapy will be applied and observation on reverse effect of cisplatin-resistance will be carried out. Therefore, an effective therapeutic strategy for overcoming chemotherapy-resistance of ovarian cancer will be initiated.Part one: The expression of ERCC1 gene in ovarian cancer cell lines and
its influence on drug-resistance of ovarian cancer cell Objective: To investigate the relationship between the expression of ERCC1 gene and cisplatin-resistance in ovarian cancer cell lines. Methods: mRNA expression of ERCC1 gene in COC1, COC1/DDP, A2780 and A2780/TS of ovarian cell line was detected with RT-PCR method, gene expression changing was observed in one day after treatment of cisplatin at different concentrations.Result: 1. ERRC1 gene expression existed in four cell lines of COC1, COC1/DDP, and A2780 and A2780/TS, Expression level of ERCC1 gene in drug-resistant subtype line of COC1/DDP was significantly higher than that of sensitive COC1, and there was statistical significance (p<0. 05);Expression level of ERCC1 gene in drug-resistant subtype line of A2780/TS was slightly higher than that of sensitive A2780, however, there was no statistical significance (p>0. 05). 2. When treated with low oncentration of cisplatin, ERCC1 gene demonstrated higher expression in C0C1 and COC1/DDP cells. ERCC1 gene displayed decline status of expression, as concentration of cisplatin increased, and decline extent of C0C1/DDP cell was obviously higher than that of C0C1 cell, there was a significant difference (p<0. 05). 3. mRNA expression of ERCC1 gene in ovarian cancer cell was related with IC50 index of cisplatin, mRNA expression quantity of ERCC1 in drug-resistant cell was significantly higher than that of sensitive cell (r=0.987, p<0. 05).Conclusion: High expression of ERCC1 gene is related with cisplatin-resistance of ovarian cancer.Part two: Construction of expression vector with ERCCl-shRNA plasmid
and detection on effect of RNA interferingObjective: To construct expression vector with ERCCl-shRNA plasmid,transient transfection was used to observe the silence effect ofrecombinant plasmid on ERCC1 gene.Methods: Small RNA interfering of short hairpin of ERCC1 gene (shRNA)was designed, plasmid expression vector with CMV promoter was cloned andrecombinant plasmid was constructed. COC1/DDP cell was transfected bylipofectane, and effect of RNA interfering on ERCC1 gene expression wasobserved.Result: 1.Successful construction of plasmid expressionnShuttle-ERCCl-shRNA . rnrn , . , ,vectorr with siRNA of ERCC1 gene hairpin was made, thesequence of heterogeneous gene was proved accurate by using digestion and sequencing. 2. RT-PCR method was used to detect mRNA expression of ERCC1 gene in COC1/DDP cells within 24, 48, 72 hours after being transfectd by recombinant plasmid, results showed that recombinant plasmidDShuttle-ERCCl-shRNA ? i n ^ ? ,. ■,■,-,r could remarkably reduce expression level of intracellularERCC1 gene in 24-74 hours after transfection, compared to that of randomlised control siRNA. Down regulation of mRNA expression level in COC1/DDP cells was separately (30.37 + 5.82) %, (53.91 + 9.12) %, (56.41 + 7.06) % (p<0. 05,p<0. 01,p<0. 01) in 24,48 and 72 hours after transfection, and results showed a statistical significance. Conclusion: Expression vector with ERCCl-shRNA plasmid is successfully constructed;mRNA expression level of ERCC1 gene may be remarkably down-regulated by constructed recombinant plasmid, and RNA interfering effect is significant.
Part Three: Construction of expression vector with ERCCl-shRNArecombinant adenovirusObjective: To construct expression vector with ERCCl-shRNA recombinantadenovirus.Methods: phosphate calcium sediment method was used to tansform twoplasmid expression vectors to HEK-293 package cells;one was shuttleplasmid of CMV promoter (pshuttle-cmv) contained hairpin siRNA with DNAtemplate, another was adenovirus skeleton plasmid which had conservativegene of adenovirus. recombinant adenoviruse was achieved by homogenousrecombinance of two plasmids in HEK-293 cells.Result: Defective type of recombinant adenovirus vector was constructedby homogenous recombinance method, augmented and purifed by chloridecentrigugation after sequenceing, final yielde were generally 3X1010pfu/ml.Conclusion: Adenovirus vector Ad-CMV-ERCCl-shRNA with ERCCl-shRNA issuccessfully constructed, it is stable, and easy to purify or augment,final yielde is high.Part Four: In vitro study on silence of ERCC1 gene by adenovirus-mediatedERCCl-shRNA gene to reverse cisplatain-resistance in ovarian cancercellsObjective: In vitro study was performed to probe into the reversibleeffect of adenovirus-mediated RNA interfering on cisplatin-resistancein ovarian cancer.Methods: COC1/DDP cells were infected by direct infection method and withrecombinant adenovirus infection. mRNA and protein expression level of
infected cells were analyzed by RT-PCR and Western Blot methods. COC1/DDP cells were infected by recombinant adenovirus in vitro and combined with therapy of cisplatin, MTT method was used to study the influence of infected cells on cisplatin-reisitance, and flow cytometry was used to observe cell cycle changing and apoptosis of infected cells. Result: l.mRNA expression of ERCCl gene in COC1/DDP cells showed down regulation within 24 hours, 48 and 72 hours after transfection by recombinant adenovirus. Protein expression level of intracellular ERCCl gene was remarkably low and related with RT-PCR result.2. Drug sensitivity test showed that COC1/DDP cells increased as the infectious viral titer and the concentration of DDP enhanced, cell survival rate significantly decreased, dose dependence existed.3. Infection of adenovirus with ERCCl-shRNA and therapy of cisplatin oculd increase cell apoptosis, and G0/Gi stage predominantly appeared. Conclusion: mRNA and protein expression of ERCCl gene is remarkably down-regulated after ERCCl gene interfered by RNA. The sensitivity of drug-resistant cell in ovarian cancer to cisplatin increases.
第一部分ERCCl基因在卵巢癌细胞系中的表达及对卵巢癌细胞耐药的影响
目的:观察人卵巢癌细胞系中ERCCl基因的表达与卵巢癌顺铂耐药的关系。方法:应用RT-PCR方法检测ERCCl基因mRNA在卵巢癌细胞系COCl、COCl/DDP、A2780及A2780/TS中的表达,观察不同浓度顺铂作用24小时后基因表达的变化。结果:1、四种卵巢癌细胞系COCl、COCI/DDP、A2780及A2780/TS中均有ERCCI基因的表达,耐药亚系COCl/DDP和亲本COCl相比,前者ERCCl基因的表达量明显高于后者,统计学检验有显著差异(p<0.05);耐药亚系A2780/TS和亲本A2780相比,ERCCl基因的表达前者比后者略有升高,无统计学意义。2、不
Ovarian cancer is a malignant cancer which seriously threatens women health. Although prognosis has been improved with development of various diagnostic and therapeutic methods, 5 year of survival rate still remains 25%-30%. Since the chemotherapy is sensitive to most of malignant ovarian cancer, especially cisplatin-based chemotherapy. It has made cisplatin play a key role in treatment of ovarian cancer. However, the resistance of chemotherapy tremendously obstructs anticancer effect of chemotherapy drug, essentially, high expression of ERCC1 gene makes cisplatin resistant. Recently found RNA interfering (RNAi) is an effective technique of gene silence, it is specifically able to make target gene expression in silence, and it has been extensively used in investigation of gene function and anticancer drugs.Adenovirus-mediated RNA interfering technique which aims at inserting exogenous small hairpin interfering RNA will be used in our study. Meanwhile, the recombinant adenovirus which stable expresses ERCC1-shRNA will be constructed by gene recombinant technology, combination with cisplatin therapy will be applied and observation on reverse effect of cisplatin-resistance will be carried out. Therefore, an effective therapeutic strategy for overcoming chemotherapy-resistance of ovarian cancer will be initiated.Part one: The expression of ERCC1 gene in ovarian cancer cell lines and
its influence on drug-resistance of ovarian cancer cell Objective: To investigate the relationship between the expression of ERCC1 gene and cisplatin-resistance in ovarian cancer cell lines. Methods: mRNA expression of ERCC1 gene in COC1, COC1/DDP, A2780 and A2780/TS of ovarian cell line was detected with RT-PCR method, gene expression changing was observed in one day after treatment of cisplatin at different concentrations.Result: 1. ERRC1 gene expression existed in four cell lines of COC1, COC1/DDP, and A2780 and A2780/TS, Expression level of ERCC1 gene in drug-resistant subtype line of COC1/DDP was significantly higher than that of sensitive COC1, and there was statistical significance (p<0. 05);Expression level of ERCC1 gene in drug-resistant subtype line of A2780/TS was slightly higher than that of sensitive A2780, however, there was no statistical significance (p>0. 05). 2. When treated with low oncentration of cisplatin, ERCC1 gene demonstrated higher expression in C0C1 and COC1/DDP cells. ERCC1 gene displayed decline status of expression, as concentration of cisplatin increased, and decline extent of C0C1/DDP cell was obviously higher than that of C0C1 cell, there was a significant difference (p<0. 05). 3. mRNA expression of ERCC1 gene in ovarian cancer cell was related with IC50 index of cisplatin, mRNA expression quantity of ERCC1 in drug-resistant cell was significantly higher than that of sensitive cell (r=0.987, p<0. 05).Conclusion: High expression of ERCC1 gene is related with cisplatin-resistance of ovarian cancer.Part two: Construction of expression vector with ERCCl-shRNA plasmid
and detection on effect of RNA interferingObjective: To construct expression vector with ERCCl-shRNA plasmid,transient transfection was used to observe the silence effect ofrecombinant plasmid on ERCC1 gene.Methods: Small RNA interfering of short hairpin of ERCC1 gene (shRNA)was designed, plasmid expression vector with CMV promoter was cloned andrecombinant plasmid was constructed. COC1/DDP cell was transfected bylipofectane, and effect of RNA interfering on ERCC1 gene expression wasobserved.Result: 1.Successful construction of plasmid expressionnShuttle-ERCCl-shRNA . rnrn , . , ,vectorr with siRNA of ERCC1 gene hairpin was made, thesequence of heterogeneous gene was proved accurate by using digestion and sequencing. 2. RT-PCR method was used to detect mRNA expression of ERCC1 gene in COC1/DDP cells within 24, 48, 72 hours after being transfectd by recombinant plasmid, results showed that recombinant plasmidDShuttle-ERCCl-shRNA ? i n ^ ? ,. ■,■,-,r could remarkably reduce expression level of intracellularERCC1 gene in 24-74 hours after transfection, compared to that of randomlised control siRNA. Down regulation of mRNA expression level in COC1/DDP cells was separately (30.37 + 5.82) %, (53.91 + 9.12) %, (56.41 + 7.06) % (p<0. 05,p<0. 01,p<0. 01) in 24,48 and 72 hours after transfection, and results showed a statistical significance. Conclusion: Expression vector with ERCCl-shRNA plasmid is successfully constructed;mRNA expression level of ERCC1 gene may be remarkably down-regulated by constructed recombinant plasmid, and RNA interfering effect is significant.
Part Three: Construction of expression vector with ERCCl-shRNArecombinant adenovirusObjective: To construct expression vector with ERCCl-shRNA recombinantadenovirus.Methods: phosphate calcium sediment method was used to tansform twoplasmid expression vectors to HEK-293 package cells;one was shuttleplasmid of CMV promoter (pshuttle-cmv) contained hairpin siRNA with DNAtemplate, another was adenovirus skeleton plasmid which had conservativegene of adenovirus. recombinant adenoviruse was achieved by homogenousrecombinance of two plasmids in HEK-293 cells.Result: Defective type of recombinant adenovirus vector was constructedby homogenous recombinance method, augmented and purifed by chloridecentrigugation after sequenceing, final yielde were generally 3X1010pfu/ml.Conclusion: Adenovirus vector Ad-CMV-ERCCl-shRNA with ERCCl-shRNA issuccessfully constructed, it is stable, and easy to purify or augment,final yielde is high.Part Four: In vitro study on silence of ERCC1 gene by adenovirus-mediatedERCCl-shRNA gene to reverse cisplatain-resistance in ovarian cancercellsObjective: In vitro study was performed to probe into the reversibleeffect of adenovirus-mediated RNA interfering on cisplatin-resistancein ovarian cancer.Methods: COC1/DDP cells were infected by direct infection method and withrecombinant adenovirus infection. mRNA and protein expression level of
infected cells were analyzed by RT-PCR and Western Blot methods. COC1/DDP cells were infected by recombinant adenovirus in vitro and combined with therapy of cisplatin, MTT method was used to study the influence of infected cells on cisplatin-reisitance, and flow cytometry was used to observe cell cycle changing and apoptosis of infected cells. Result: l.mRNA expression of ERCCl gene in COC1/DDP cells showed down regulation within 24 hours, 48 and 72 hours after transfection by recombinant adenovirus. Protein expression level of intracellular ERCCl gene was remarkably low and related with RT-PCR result.2. Drug sensitivity test showed that COC1/DDP cells increased as the infectious viral titer and the concentration of DDP enhanced, cell survival rate significantly decreased, dose dependence existed.3. Infection of adenovirus with ERCCl-shRNA and therapy of cisplatin oculd increase cell apoptosis, and G0/Gi stage predominantly appeared. Conclusion: mRNA and protein expression of ERCCl gene is remarkably down-regulated after ERCCl gene interfered by RNA. The sensitivity of drug-resistant cell in ovarian cancer to cisplatin increases.
引文
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