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

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"

[1] Hu N, Feng C, Jiang Y , et al. Regulative effect of Mir-205 on osteogenic differentiation of bone mesenchymal stem cells (BMMSCs): possible role of SATB2/Runx2 and ERK/MAPK pathway[J]. Int J Mol Sci, 2015,16(5):10491-10506.
doi: 10.3390/ijms160510491 pmid: 4463658
[2] Ichijima T, Matsuzaka K, Tonogi M , et al. Osteogenic differences in cultured rat periosteal cells under hypoxic and normal conditions[J]. Exp Ther Med, 2012,3(2):165-170.
doi: 10.3892/etm.2011.393 pmid: 22969863
[3] Heo JH, Choi JH, Kim IR , et al. Combined treatment with low-level laser and rhBMP-2 promotes differentiation and mineralization of osteoblastic cells under hypoxic stress[J]. Tissue Eng Regen Med, 2018,5(6):793-801.
doi: 10.1007/s13770-018-0167-1
[4] Chen XY, Liu Y, Ding WH , et al. Mechanical stretch-induced osteogenic differentiation of human jaw bone marrow mesenchymal stem cells (hJBMMSCs) via inhibition of the NF-kappa B pathway[J]. Cell Death Dis, 2018,9(2):207.
doi: 10.1038/s41419-018-0279-5 pmid: 29434225
[5] Yao Q, Liu Y, Selvaratnam B , et al. Mesoporous silicate nanoparticles/3D nanofibrous scaffold-mediated dual-drug delivery for bone tissue engineering[J]. J Control Release, 2018,279:69-78.
doi: 10.1016/j.jconrel.2018.04.011 pmid: 29649529
[6] Will LA . Orthodontic tooth movement: a historic prospective[J]. Front Oral Biol, 2016,18:46-55.
doi: 10.1159/000351899 pmid: 26599117
[7] 邓正炜, 张亚东, 赵世昌 . 组织工程骨激活低氧诱导因子HIF-1α促进骨修复[J]. 实用骨科杂志, 2018,24(10):917-921.
[8] Wang Y, Wan C, Deng L , et al. The hypoxia-inducible factor α pathway couples angiogenesis to osteogenesis during skeletal development[J]. J Clin Investig, 2007,117(6):1616-1626.
doi: 10.1172/JCI31581 pmid: 1878533
[9] Yang DC, Yang MH, Tsai CC , et al. Hypoxia inhibits osteogenesis in human mesenchymal stem cells through direct regulation of RUNX2 by TWIST[J]. PLoS One, 2011,6(9):e23965.
doi: 10.1371/journal.pone.0023965 pmid: 21931630
[10] Carroll SF, Buckley CT, Kelly DJ . Cyclic tensile strain can play a role in directing both intramembranous and endochondral ossification of mesenchymal stem cells[J]. Front Bioeng Biotechnol, 2017,5:73.
doi: 10.3389/fbioe.2017.00073 pmid: 5712005
[11] Boink MA , Van Den Broek LJ, Roffel SA, et al. Different wound healing properties of dermis, adipose, and gingiva mesenchymal stromal cells[J]. Wound Repair Regen, 2016,24(1):100-109.
doi: 10.1111/wrr.12380 pmid: 26542883
[1] Lin YUAN, Jun QIAN, Zhengyi YANG, Han WANG, Wucheng GUO, Jieli CHENG, Jingjing SONG, Enliang HE, Yi ZHANG. Comparison of osteogenic differentiation abilities of mesenchymal stem cells from different sources of hBMSCs [J]. Journal of Prevention and Treatment for Stomatological Diseases, 2017, 25(9): 554-559.
Viewed
Full text


Abstract

Cited

  Shared   
  Discussed   
[1] Hong-chang LAI,Jun-yu SHI. Maxillary sinus floor elevation[J]. Journal of Prevention and Treatment for Stomatological Diseases, 2017, 25(1): 8 -12 .
[2] Pin ZHOU, Yang-fei LI. MRI study of temporomandibular joint disc position in asymptomatic volunteers[J]. Journal of Prevention and Treatment for Stomatological Diseases, 2017, 25(4): 239 -244 .
[3] Xinxin XIA, Fang FANG, Lijuan CHENG. Shaping ability of Pathfile and WaveOne in simulated root canals[J]. Journal of Prevention and Treatment for Stomatological Diseases, 2017, 25(6): 365 -368 .
[4] Yuanhong LI, Xinyi FANG, Yu QIU, Lei CHENG. Experimental study on the effects of green tea on salivary flow rate and pH value[J]. Journal of Prevention and Treatment for Stomatological Diseases, 2017, 25(9): 560 -564 .
[5] Chengzhang LI. Masticatory muscles in occlusion[J]. Journal of Prevention and Treatment for Stomatological Diseases, 2017, 25(12): 755 -760 .
[6] . [J]. Journal of Prevention and Treatment for Stomatological Diseases, 2018, 26(1): 1 .
[7] Zhirong WU, Shiguang Huang. Research progress on the etiology, clinical examination and treatment of peri-implantitis[J]. Journal of Prevention and Treatment for Stomatological Diseases, 2018, 26(6): 401 -405 .
[8] Xiaowu YAO, Shisheng CHEN, Zizheng LU, Minxiao LIN. Clinical report and literature review on the amyloidosis of salivary glands[J]. Journal of Prevention and Treatment for Stomatological Diseases, 2018, 26(8): 533 -536 .
[9] Lan LIAO, Lijun ZENG. Updated research on digitalization in aesthetic restoration[J]. Journal of Prevention and Treatment for Stomatological Diseases, 2018, 26(7): 409 -414 .
[10] Yu LU, Chengxia LIU, Zhongjun LIU. Role of TRAF6 in inflammatory responses of human osteoblast-like cells with Enterococcusfaecalis[J]. Journal of Prevention and Treatment for Stomatological Diseases, 2017, 25(7): 420 -425 .
This work is licensed under a Creative Commons Attribution 3.0 License.