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目的构建micoRNA-205(miR-205)慢病毒表达载体,感染乳腺癌细胞MB231,建立稳定表达miR-205的MB231细胞株,观察其增殖能力的变化。方法酶切慢病毒载体GV369,设计并合成miR-205引物,通过PCR扩增目的基因连接到慢病毒载体上。对重组质粒双酶切鉴定,进行慢病毒hsa-miR-205的包装和滴度测定。用构建好的慢病毒感染MB231细胞,定量PCR检测细胞中miR-205表达水平的变化,MTT和划痕实验观察过表达miR-205后MB231细胞增殖和迁移能力的变化。结果测序显示,目的基因连接慢病毒载体成功。慢病毒稳定感染了MB231,定量PCR结果显示,感染hsa-miR-205的MB231中miR-205表达明显提高,MTT和划痕实验结果显示感染后MB231的细胞增殖和迁移能力受到抑制。结论成功构建了miR-205慢病毒表达载体,并建立了稳定表达miR-205的细胞株MB231,显示其可能负调控乳腺癌细胞的恶性生物学行为,为后期进一步研究miR-205的功能和机制奠定了基础。
Objective To construct a micoRNA-205 (miR-205) lentivirus expression vector and to infect breast cancer cell MB231, establish a MB231 cell line stably expressing miR-205, and observe the changes in its proliferation ability. Methods The lentiviral vector GV369 was digested and the miR-205 primers were designed and synthesized. The target gene was amplified by PCR and ligated into lentivirus vector. For the identification of double-enzyme digestion of recombinant plasmids, the lentivirus hsa-miR-205 was packaged and titrated. MB231 cells were infected with the established lentivirus, and the expression of miR-205 in cells was detected by quantitative PCR. The proliferation and migration of MB231 cells after overexpression of miR-205 were observed by MTT and scratch experiments. The sequencing of the results showed that the target gene was successfully ligated to the lentivirus vector. Lentiviruses were stably infected with MB231. Quantitative PCR showed that the expression of miR-205 in MB231 infected with hsa-miR-205 was significantly increased. MTT and scratch test results showed that the cell proliferation and migration of MB231 were inhibited after infection. Conclusion The miR-205 lentiviral expression vector was successfully constructed, and the cell line MB231 stably expressing miR-205 was established, indicating that it may negatively regulate the malignant biological behavior of breast cancer cells, and further study the function and mechanism of miR-205 for the later period. Foundation.