【作者】 李智敏；

【导师】 王泽华；

【作者基本信息】 华中科技大学 ， 妇产科学， 2010， 博士

【摘要】 目的：研究卵巢癌细胞及乳腺癌细胞中hsa-miR-27a和hsa-miR-451的表达差异。方法：用浓度递增法建立卵巢癌耐紫杉醇细胞系A2780/Taxol; Western blot检测卵巢癌和乳腺癌细胞中P-糖蛋白(P-glycoprotein, P-gp)的表达；Stem-loop real-time PCR检测卵巢癌耐紫杉醇细胞A2780／Taxol和亲本细胞A2780以及乳腺癌耐阿霉素细胞MCF-7/ADM和亲本细胞MCF-7中hsa-miR-27a和hsa-miR-451的表达。结果：成功建立了卵巢癌耐紫杉醇细胞株A2780/Taxol; P-gp在卵巢癌耐紫杉醇细胞A2780/Taxol和乳腺癌耐阿霉素细胞MCF-7/ADM中明显高表达,在卵巢癌和乳腺癌亲本细胞A2780和MCF-7中均未检测到P-gp蛋白的表达；miR-27a在A2780/Taxol细胞中高表达,与A2780细胞相比,表达增高2.2±0.30倍,差异有统计学意义(p<0.05)。在A2780和A2780/Taxol细胞中未检测出miR-451表达的差异。miR-451在MCF-7/ADM细胞中低表达,与MCF-7细胞相比,表达降低16±0.21倍,差异有统计学意义(p<0.05)。在MCF-7和MCF-7/ADM中未检测出miR-27a表达的差异。结论：在P-gp高表达的卵巢癌耐紫杉醇细胞和乳腺癌耐阿霉素细胞中,miR-27a和miR-451分别特异性的表达异常。目的：研究hsa-miR-27a与卵巢癌耐紫杉醇细胞耐药的关系。方法：利用脂质体Lipofectamine 2000将成熟miR-27a的模拟物mimtcs、阻遏物inhibitors及阴性对照negative control RNA (NC)转染A2780和A2780/Taxol细胞；Real-time PCR技术检测细胞多药耐药-1(MDR1)基因表达；蛋白印迹法检测转染后A2780/Taxol细胞P-gp和同源结构域相关的蛋白激酶2(homeodomain-interacting protein kinase 2, HIPK2)蛋白表达；MTT检测细胞增殖；Annexin-V FITC/PI双标法流式细胞术检测细胞凋亡率；流式细胞术检测细胞内罗丹明123(Rh-123)蓄积量。结果：1. A2780/Taxol细胞转染miR-27a inhibitors后(1)MDR1 mRNA表达明显下降,与转染NC组相比,表达下降(39±0.14)%,差异有统计学意义(P<0.05)；(2)P-gp蛋白相对表达量((26±5.3)%)与转染NC组的P-gp蛋白相对表达量((43±6.7)%)比较,下降39%,差异有统计学意义(P<0.05)；(3)HIPK2蛋白相对表达量((30±5.9)%)与转染NC组的HIPK2蛋白相对表达量((19±3.8)%)比较,增高59%,差异有统计学意义(P<0.05)；(4)对紫杉醇的敏感性增加,半数抑制浓度(IC50)为0.53μM,与转染NC组IC50 6.8μM相比,差异有统计学意义(P<0.05)；(5)细胞凋亡率明显升高,凋亡率达(32.5±3.6)%,与转染NC组凋亡率(5.6±2.1)%比较,差异有统计学意义(P<0.05)；(6)细胞内Rh-123荧光强度增高为5.10±0.35,与转染前A2780/Taxol细胞的2.95±0.39和转染NC的A2780/Taxol细胞的3.30±0.45相比,均有显著性差异(P<0.05)。2.转染miR-27a mimics后,(1)A2780细胞的MDR1 mRNA表达升高,与转染NC组相比,升高(121±0.11)%,差异有统计学意义(P<0.05)；(2)A2780细胞对紫杉醇的敏感性下降,IC50为0.2μM,与转染NC组IC50 0.06μM相比,差异有统计学意义(P<0.05)；(3) A2780/Taxol细胞的HIPK2蛋白相对表达量((8.1±4.9)%)与转染NC组HIPK2蛋白相对表达量((19±3.8)%)比较,下降53%,差异有统计学意义(P<0.05)。结论：miR-27a在卵巢癌耐紫杉醇细胞中高表达,可能通过调控靶基因HIPK2,间接调节MDR1/P-gp的表达和功能,参与耐药。转染miR-27a inhibitors后,可以下调P-gp表达和功能,增加A2780/Taxol细胞对紫杉醇的敏感性,部分逆转耐药。目的：研究hsa-miR-451与乳腺癌耐阿霉素细胞耐药关系的研究。方法：利用脂质体Lipofectamine 2000将成熟miR-451的模拟物mimics及阴性对照negative control RNA (NC)转染MCF-7/ADM细胞；Real-time PCR技术和western blot技术分别检测转染后MCF-7/ADM细胞的MDR1 mRNA和P-gp蛋白表达；MTT检测细胞增殖；流式细胞术检测细胞内阿霉素蓄积量。结果：MCF-7/ADM细胞转染1miR-451 mimics后(1)MDR1 mRNA表达明显下降,与转染NC组细胞相比,表达下降(65±12)%,差异有显著性(P<0.05)；(2)P-gp蛋白相对表达量((31±19)%)与转染NC组细胞P-gp蛋白相对表达量((83±12)%)相比,下降62%,差异有显著性(P<0.05)；(3)对阿霉素的敏感性增加,IC50为4.61μM,与转染NC组细胞IC5026μM相比,有显著性差异(P<0.05)；(4)细胞内的阿霉素荧光强度增加为28.98±2.9,与转染NC组细胞的11.64±2.6相比有显著性差异(P<0.05)。结论：miR-451在乳腺癌耐阿霉素细胞MCF-7/ADM中低表达,它可能通过负性调控靶基因P-gp蛋白参与乳腺癌耐阿霉素的发生。转染miR-451 mimics后,可以增加MCF-7/ADM细胞对阿霉素的敏感性,部分逆转耐药。更多还原

【Abstract】 Objective To investigate the expression of miR-27a and miR-451 in ovarian cancer and breast cancer cells.Methods A2780/Taxol cells were established using stepwise selection. The expression of P-gp protein levels of ovarian cancer and breast cancer cells were measured by western blot. Stem-loop real-time PCR was used to detect expression of miR-27a and miR-451 in ovarian cancer and in breast cancer cells.Results A2780/Taxol cell line was established successfully. The expression levels of P-gp were high in A2780/Taxol and MCF-7/ADM cells and it did not detected in A2780 and MCF-7 cells. The expression of miR-27a was an average of (2.2±0.30)-fold higher in A2780/Taxol cells than in A2780 cells, with a significant difference between the two groups (P<0.05).The expression of miR-451 was an average of (16±0.21)-fold lesser in MCF-7/ADM cells than in MCF-7 cells, with a significant difference between the two groups (P<0.05).Conclusion The expressions of miR-27a and miR-451 were deregulation in A2780/Taxol and MCF-7/ADM cells respectively. Objective To investigate the relationship between miR-27a and drug resistant in paclitaxel-resistant ovarian cancer cells.Methods The A2780 and A2780/Taxol cells were transfected with the mimics or inhibitors of miR-27a or negative control RNA (NC) by Lipofectamine 2000. The expressions of MDR1 mRNA, P-gp and HIPK2 protein levels were measured by real-time PCR and western blot respectively. MTT was used to analyze drug sensitivity. Apoptosis analysis and the fluorescence of remaining Rh-123 were measured by fluorescence activated cell sorter (FACS).Results (1) A2780/Taxol cells transfection with inhibitors of miR-27a showed that the levels of MDR1 mRNA was decreased by (39±0.14)%, P-gp protein level((26±5.3)%) decreased than the NC group ((43±6.7)%), HIPK2 protein level ((30±5.9)%) increased than the NC group ((19±3.8)%), the IC50 (0.53μM) was less than the NC group (6.8μM), apoptosis rate ((32.5±3.6)%) was higher than the NC group ((5.6±2.1)%), the fluorescence intensity of Rh-123 (5.10±0.35) was increased than the NC group (3.30±0.45), and there was a significant difference between two groups (P<0.05). (2) Transfection of A2780 cells with mimics of miR-27a led to increase MDR1 mRNA expression by (121±0.11)% and decrease the sensitivity to paclitaxel (IC50 0.2μM vs.0.06μM). (3) HIPK2 protein level ((8.1±4.9)%) in A2780/Taxol cells management with mimics of miR-27a was decreased than the NC group ((19±3.8)%). There was a significant difference between two groups (P<0.05).Conclusion The expression of miR-27a is upregulated in A2780/Taxol cells, and miR-27a may regulate MDR1/P-gp expression by targeting HIPK2. Transfection with miR-27a inhibitors can downregulate the P-gp expression and activation, and reverse drug resistance in A2780/Taxol cells.Objective To study the role of miR-451 in the development of adriamycin-resistance in breast cancer cells.Methods The mimics of miR-451 and negative control (NC) were transfected into MCF-7/ADM cells by Lipofectamine 2000. The expression levels of MDR1 mRNA and P-gp protein were examined using real time quantitative PCR and western blot respectively. Half-inhibitory concentration (IC50) of doxorubicin was determined by MTT method and the intracellular accumulation of doxorubicin was measured by FACS.Results Transfection of MCF-7/ADM cells with mimics of miR-451 showed that expression of MDR1 mRNA was decreased by (65±12)%, P-gp protein ((31±19)%) was less than the NC group ((83±12)%), the sensitivity of cells to adriamycin enhanced and the IC50 of adriamycin (4.61μM) was less than the NC group (26μM), and the fluorescence intensity of intracellular doxorubicin (28.98±2.9) was increase than the NC group (11.64±2.6). There was a significant difference between two groups (P<0.05).Conclusion The deregulation of miR-451 may be involved in the development of adriamycin-resistance, at least in part, by negatively regulating expression and activation of MDR1/P-gp in MCF-7/ADM cells.更多还原