整合素β1基因shRNA慢病毒载体构建及其对乳腺癌他莫昔芬耐药细胞迁移和侵袭能力的影响

Construction of lentivirus-mediated shRNA vector targeting the human ITGβ1 gene and indentification of its effect on migration and invasion of tamoxifen-resistant MCF-7

摘要:

目的：构建整合素β1（integrin β1，ITGβ1）基因干扰慢病毒载体，并研究ITGβ1对他莫昔芬耐药乳腺癌MCF-7细胞（Tamoxifen-resistant MCF-7，MCF-7R）的迁移和侵袭能力的影响。方法：针对ITGβ1靶序列设计合成4条shRNA序列及1条阴性对照序列NC，构建ITGβ1RNAi慢病毒载体及对照病毒载体，并分别进行病毒包装，通过内源性筛靶，选取干扰效果最好的组病毒感染MCF-7R细胞，Western blot检测ITGβ1蛋白的表达水平；Transwell实验检测细胞迁移和侵袭能力。结果：成功构建4条慢病毒干扰重组质粒，并分别包装成病毒，通过内源性筛靶挑选出干扰效果最佳的慢病毒组，感染MCF-7R细胞后ITGβ1基因的蛋白表达量较NC组明显降低（P=0.000），且细胞的迁移和侵袭能力明显弱于NC组（P=0.000）。结论：慢病毒介导的shRNA可有效地干扰MCF-7R细胞中ITGβ1的表达，并且抑制MCF-7R细胞的迁移和侵袭能力。

Abstract:

Objective：To construct lentivirus vector targeting integrin β1（ITGβ1） gene and to study its effect on migration and inva－sion of Tamoxifen-resistant MCF-7（MCF-7R）. Methods：Four shRNA sequences targeting ITGβ1 gene and one negative control se－quence were designed，and were cloned into lentivirus vector，respectively. Recombinant lentivirus and control were extracted after transfecting 293T with the recombinant vector and helper vectors. The lentivirus with the best interfering effect was determined by real-time PCR and was chosen to infect MCF-7R. The expression of ITGβ1 gene was determined by Western blot. Transwell assay was used to detect the difference of migration and invasion. Results：Four recombinant lentivirus vectors were successfully constructed and the packaged lentivirus with the best interfering effect was used to infect MCF-7R. Western blot results showed that the protein expression of ITGβ1 was significantly reduced in MCF-7R（P=0.000） and its migration and invasion abilities were markedly suppressed（P=0.000）. Conclusion：Lentivirus vector targeting ITGβ1 gene can efficiently suppress the expression of ITGβ1 in MCF-7R，thereby leading to decreased migration and invasiveness abilities of MCF-7R