Effects of silencing the HIF-1α gene on the expression of BSP and osterix in rat BMMSCs under tension

Abstract

Objective To explore the effect of hypoxia inducible factor 1α (HIF⁃1α) gene silencing in rat bone mar⁃ row mesenchymal stem cells (BMMSCs) under mechanical distraction on the expression of bone sialoprotein (BSP) and osterix and to provide a new idea for repairing bone defects with BMMSCs. Methods The shRNA sequence was de⁃signed according to the rat HIF⁃1α gene, and the pGMLV⁃SC1RNAi lentiviral vector was cloned after PCR amplifica⁃ tion. After screening positive clones and identifying competent transformed cells by sequencing, 293T cells were pack⁃ aged and titered, rat BMMSCs were transfected and cultured in vitro. Clones with stably silenced HIF⁃1α expression were screened by inverted fluorescence microscopy. The RNAi response experiment was divided into four groups: the blank control group, the HIF⁃1α shRNA group, the negative control group, and the response group. Western blot was used to detect the expression of HIF⁃1α protein in the four groups to verify the response of the target genes and exclude off⁃target effects. A Flexcell FX⁃5000T cell stress loading system was used to intervene in the mechanical stretch of the cells. qRT⁃PCR and Western blot were used to detect the expression of BSP and osterix in the blank control group, HIF⁃ 1α shRNA group, and negative control group. Results The HIF ⁃ 1α shRNA lentiviral vector was successfully con⁃ structed. The results of the RNAi response showed no significant difference in the expression of HIF⁃1α between the re⁃ sponse and the blank control group (P > 0.05). The recombinant lentivirus could effectively silence HIF ⁃ 1α in BMMSCs. After mechanical distraction of the BMMSCs, compared with the blank and negative control groups, the HIF⁃ 1α shRNA group showed significantly increased mRNA and protein expression of the bone⁃related factors BSP and os⁃ terix (P 0.05). Conclusion Silencing HIF⁃1α in BMMSCs under mechanical distrac⁃ tion can promote the expression of BSP and osterix.