β-thalassemia for translating human β-globin gene and RNA interference of α-globin gene expression by lentivirus vectors

Objective: According to the characteristics of alpha/beta-globin gene imbalance caused by alpha-globin gene accumulation in beta-thalassemia, we designed two lentiviral vectors, that is, while we used the lentiviral to mediate full length human beta-globin gene, we introduced the interference fragment of lentiviral alpha-globin gene to adjust the alpha/beta chain ratio by increasing the expression of beta-globin and decreasing the expression of alpha-globin. Methods: We gained lentiviral vectors LV-beta-globin by extracting DNA of beta-globin expresser. According to the design principles of RNA interference sequence, the optimal dynamic parameters of RNA interference target sequence are selected based on the design experience and software evaluation. After lentivirals were stably transfected into K562 cells, the total RNA was extracted, and the expression of m RNA of alpha-(HbA2) and beta-(HBB) globin gene in K562 cells was detected by PCR real-time. Western blotting was used to detect the expression of alhpa and beta-globin. Results: beta-globin gene was amplified to specific band of 227 bp by RT-PCR, and the expression of RT-PCR alpha-globin gene was amplificated to specific band of 85 bp as well. 2(-Delta Delta Ct) value of beta-globin gene mRNA was 4.080 +/- 0.078 and 2(-Delta Delta Ct) value of alpha-globin gene mRNA was 0.274 +/- 0.023. Results of western blotting test indicated that in the stably transfected K562 cells, the synthesis of human beta-globin was significantly higher than that of the blank group and negative control group and the synthesis of alpha-globin was significantly lower than that of the blank group and negative control group. Western blotting showed that the grey level of beta-globin was 1.063 +/- 0.082, and the grey level of alpha-globin was 0.327 +/- 0.042, with statistical significance. Conclusions: This study successfully transfected beta-globin gene and alpha-globin gene siRNA into K562 cell. After lentiviral transfection, the levels of beta-globin gene RNA and protein were significantly higher than the control group.