溶酶体相关膜蛋白2AshRNA对乳腺癌细胞株紫杉醇耐药的影响  

The effect of LAMP2A shRNA on the resistance of breast cancer cells to paclitaxel

作　　者：韩起[1] 陈莎[1] 杨明珍[1] 张竹君[1] 陈安[1] 胡川闽[1] 李淑慧[1] 

机构地区：[1]第三军医大学西南医院医学检验系临床生物化学教研室

出　　处：《细胞与分子免疫学杂志》2014年第4期351-354,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金(81272909)

摘　　要：目的制备针对人溶酶体相关膜蛋白2A(LAMP2A)的shRNA重组慢病毒,获得稳定低表达LAMP2A的MDA-MB-231乳腺癌细胞株,观察LAMP2A分子下调对乳腺癌细胞紫杉醇耐药性的影响。方法通过对LAMP2A的mRNA序列分析,设计了4条shRNA,通过基因重组技术构建pGLV-EGFP-shRNA慢病毒表达质粒,并测序鉴定。将构建的表达质粒和包装质粒共转染人胚肾HEK293T细胞包装产生慢病毒,测定病毒滴度。将构建的shRNA慢病毒表达载体感染人乳腺癌MDA-MB-231细胞,嘌呤霉素筛选获得稳定细胞株后,Western blot法检测LAMP2A表达,验证蛋白表达抑制效果。采用1 nmol/L、10 nmol/L、100nmol/L的紫杉醇处理LAMP2A低表达乳腺癌细胞株后,用MTT法观察LAMP2A低表达组和对照组之间增殖效率的差异。结果测序结果显示重组慢病毒质粒构建正确,病毒滴度达2×108TU/mL。乳腺癌稳定细胞株中LAMP2A蛋白表达量显著降低。在10 nmol/L、100 nmol/L紫杉醇处理后,LAMP2A低表达乳腺癌细胞株对紫杉醇耐药性明显降低(P<0.05)。结论成功构建了人的LAMP2A基因shRNA慢病毒载体,获得了稳定低表达LAMP2A的乳腺癌细胞株,证实LAMP2A下调能够降低乳腺癌细胞株对紫杉醇的耐药性。Objective To prepare a recombinant lentiviral carring human lysosorne-associated membrane protein type 2A （LAMP2A） gene shRNA, establish MDA-MB-231 cell line with a low expression of LAMP2A and observe the change in cell resistance to paclitaxel. Methods Four shRNAs were designed according to the sequencing analysis of LAMP2A mRNA. The pGLV-EGFP-shRNA lentiviral vector was established by gene recombination technology and was confirmed by DNA sequencing. The lentiviral vector and the packaging vector were used to cotransfect the HEK293T cells to obtain the lentivirus against LAMP2A mRNA, and the titer of the virus was determined. The constructed shRNA lentivirus was applied to infect human breast cancer cell line MDA-MB-231, and then the cells were screened with puromycin for two weeks. The inhibitive efficacy of the shRNA on the LAMP2A protein was determined by Western blotting. After the breast cancer cell line with a low expression of LAMP2A was treated with three different concentrations of paclitaxel （l, 10, 100 nmol/L）, M-I-r assay was performed to observe the difference in the proliferation ability between the control group and the low LAMP2A expression groups. Results DNA sequencing revealed that the recombinant lentiviral plasmid was correctly constructed with the virus titer reaching 2 x 108 TU/mL. The LAMP2A protein expression in the obtained breast cancer cell line dropped drastically. After the treatment with paclitaxel at 10 and 100 nmol/L respectively, the drug resistance of cells with a low expression of LAMP2A was notably weaker than that of the control group （ P 〈 0.05 ）. Conclusion The recombinant shRNA lentiviral vector against human LAMP2A gene was successfully constructed, and the breast cancer cell line MDA-MB-23! transfected with it stably expressed a low level of LAMP2A. It was proved that the down-regulation of LAMP2A could reduce the resistance of breast cancer cells to paclitaxel.

关 键 词：溶酶体相关膜蛋白2A 小发夹RNA 慢病毒载体 紫杉醇 

分 类 号：Q811.4[生物学—生物工程] R392.11[医药卫生—免疫学]