Journal of Prevention and Treatment for Stomatological Diseases ›› 2019, Vol. 27 ›› Issue (5): 287-292.doi: 10.12016/j.issn.2096-1456.2019.05.003

• Basic Study • Previous Articles     Next Articles

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

LIU Ying1,YANG Jing1,LI Yazhen1,YAN Xiao2,ZHANG Qiang2,REN Dapeng2,YANG Fang1,4,YUAN Xiao1,2,GUO Qingyuan1,3,4,()   

  1. 1. Stomatology College of Qingdao University, Qingdao 266071, China
    2. No.2 Department of Orthodontics, The Affiliated Hospital of Qingdao University, Qingdao 266003, China
    3. Institute of Stomatology, Chinese PLA General Hospital, Beijing 100853, China
    4. Department of Stomatology, The Affiliated Qingdao Municipal Hospital,Qingdao 266071, China
  • Received:2018-10-19 Revised:2018-11-06 Online:2019-05-20 Published:2019-05-20
  • Contact: Qingyuan GUO E-mail:guoqingyuanqd@163.com

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Abstract:

Objective To explore the effect of hypoxia inducible factor 1α (HIF-1α) gene silencing in rat bone marrow 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 designed according to the rat HIF-1α gene, and the pGMLV-SC1RNAi lentiviral vector was cloned after PCR amplification. After screening positive clones and identifying competent transformed cells by sequencing, 293T cells were packaged 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 constructed. The results of the RNAi response showed no significant difference in the expression of HIF-1α between the response 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 osterix (P < 0.05); there was no significant difference in the mRNA and protein expression of BSP or osterix between the blank and negative control groups (P > 0.05).Conclusion Silencing HIF-1α in BMMSCs under mechanical distraction can promote the expression of BSP and osterix.

Key words: Hypoxia-inducible factor-1α, Lentivirus vector, Gene silencing, Bone marrow mesenchymal stem cells, Mechanical stretch, Bone sialoprotein, Osterix

CLC Number: 

  • R78

Table 1

Nucleotide sequences of HIF-1α shRNA and HIF-1α NC"

引物 序列 （5′→3′）
shRNA-F
shRNA-R
NC-F
NC-R		 5′-GATCCGCAGACCCAGTTACAGAAACCTTCAAGAGAGGTTTCTGTAACTGGGTCTGCTTTTTTG-3′
5′-AATTCAAAAAAGCAGACCCAGTTACAGAAACCTCTCTTGAAGGTTTCTGTAACTGGGTCTGCG-3′
5′-GATCCGTTCTCCGAACGTGTCACGTTTCAAGAGAACGTGACACGTTCGGAGAACTTTTTTACGCGTG-3′
5′-AATTCACGCGTAAAAAAGTTCTCCGAACGTGTCACGTTCTCTTGAAACGTGACACGTTCGGAGAACG-3′

Table 2

Nucleotide sequences of the primers used for qRT-PCR"

引物 序列（5′→3′） 片段大小（bp）
BSP-F
BSP-R
Osterix-F
Osterix-R
GAPDH-F
GAPDH-R		 5′-ATGGAGACGGCGATAGTTCC-3′
5′-CTAGCTGTTACACCCGAGAGT-3′
5′-ACCCCAAGATGTCTATAAGCCC-3′
5′-CGCTCTAGCTCCTGACAGTTG-3′
5′-AGGTCGGTGTGAACGGATTTG-3′
5′-TGTAGACCATGTAGTTGAGGTCA-3′		 148
148
168
168
123
123

Figure 1

Sequencing screenshot of rat HIF-1α shRNA lentiviral vector"

Figure 2

Immunofluorescence microscopy observations at 96 hours after lentiviral transduction of 293T cells × 100"

Figure 3

Western blot analysis of gene silencing effect of HIF-1α"

Figure 4

Western blot analysis of off-target effects of shRNA"

Figure 5

mRNA and protein expression of osteogenic related genes in rat BMMSCs of 3 groups under mechanical tension"

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