稳定表达HMGA2的人乳腺癌细胞株 MDA-MB-231 构建.

Objective To establish the human breast MDA-MB-231 cells stably expressing HMGA2 protein. Methods Total RNA of MDA-MB-231 cells in the logarithmic phase were extracted, and cDNA of HMGA2 gene was reversely tran­scribed. The recombinant pLVX-HMGA2 expression vector was acquired by inserting the target fragments which were ac­quired by PCR technology into the lentiviral expression plasmid pLVX-IRES-Puro. The forward and reverse primers, contai­ning EcoR1 and BamH1 restriction enzyme sites, were designed according to their CDS sequences. The recombinant lenti- virus carrying HMGA2 gene was obtained by co-transfecting HEK293T cells with the packaging plasmid，and the culture supernatant containing the lentiviral particles was collected. The recombinant plasmid was identified by bacterial colonies PCR，double restriction enzyme digestion，plasmid PCR，and sequencing comparison. MDA-MB-231 cells were divided in­to two groups. The cells in the experimental group were infected by the lentivirus carrying HMGA2 gene，simultaneously， the control group was infected by the lentivirus carrying only expression vector. Puromycin (1 pg/mL) was used to screen out the infected cells after 48 hours so as to obtain a cell line for stable expression. The mRNA and protein expression of HMGA2 was detected by Western blotting and real-time fluorescent quantitative PCR. Results The relative expression levels of HMGA2 mRNA in the observation group and the control group were 10 273.93 ±4 290. 77 and 1.18 ±0.81，re­spectively， and significant difference was found between these two groups (P < 0. 05 ). The relative expression levels of HMGA2 protein in the observation group and the control group were 1.570 ±0.080 and 0. 110 ±0.002，and the difference between the two groups was statistically significant ( P <0. 05 ) . Conclusion The breast cancer cell line MDA-MB-231 stably expressing HMGA2 is successfully established. [ABSTRACT FROM AUTHOR]