Effect of knocking down Piezo1 mechanically sensitive protein on migration of MC3T3-E1 osteoblast cells_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

YAN Liang , JIANG Jin , MA Chongwen , LI Rui ,  XIA Yayi

Department of Orthopedics, Gansu Key Laboratory of Orthopaedics, Lanzhou University Second Hospital, Lanzhou Gansu, 730000, P.R.China;

Corresponding author：

XIA Yayi, Email: xiayayi@126.com

Keywords：

Piezo1; small interfering RNA; MC3T3-E1 osteoblast cells; cell migration

DOI：

10.7507/1002-1892.201806121

Video：

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Objective To discuss the effect of Piezo1 mechanically sensitive protein in migration process of mouse MC3T3-E1 osteoblast cells.Methods The 5th-10th generation mouse MC3T3-E1 osteoblasts were divided into Piezo1-small interfering RNA (siRNA) transfection group (group A), negative control group (group B), and blank control group (group C). Piezo1-siRNA or negative control siRNA was transfected into mouse MC3T3-E1 osteoblasts by siRNA transfection reagent, respectively; group C was only added with siRNA transfection reagent; and the cell morphology was observed under inverted phase contrast microscope and fluorescence microscope, and the transfection efficiency was calculated. The expression of Piezo1 protein was detected by immunofluorescence staining and Western blot. Transwell cell migration assay and cell scratch assay were used to detect the migration of MC3T3-E1 osteoblasts after Piezo1-siRNA transfection.Results After 48 hours of transfection, group A showed a slight increase in cell volume and mutant growth, but cell colonies decreased, suspension cells increased and cell fragments increased when compared with untransfected cells. Under fluorescence microscope, green fluorescence was observed in MC3T3-E1 osteoblasts of group B, and the transfection efficiency was 68.56%±4.12%. Immunofluorescence staining and Western blot results showed that the expression level of Piezo1 protein in group A was significantly lower than that in groups B and C (P<0.05); there was no significant difference between group B and group C (P>0.05). Transwell cell migration assay and cell scratch assay showed that the number of cells per hole and the scratch healing rate of cells cultured for 1-4 days in group A were significantly lower than those in groups B and C (P<0.05); there was no significant difference between group B and group C (P>0.05).Conclusion Piezo1 knocked down by siRNA can inhibit the migration ability of MC3T3-E1 osteoblast cells.

Citation： YAN Liang, JIANG Jin, MA Chongwen, LI Rui, XIA Yayi. Effect of knocking down Piezo1 mechanically sensitive protein on migration of MC3T3-E1 osteoblast cells. Chinese Journal of Reparative and Reconstructive Surgery, 2019, 33(1): 28-34. doi: 10.7507/1002-1892.201806121 Copy

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8.	Coste B, Mathur J, Schmidt M, et al. Piezo1 and Piezo2 are essential components of distinct mechanically activated cation channels. Science, 2010, 330(6000): 55-60.
9.	Sugimoto A, Miyazaki A, Kawarabayashi K, et al. Piezo type mechanosensitive ion channel component 1 functions as a regulator of the cell fate determination of mesenchymal stem cells. Sci Rep, 2017, 7(1): 17696.
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13.	Vishnuprasad CN, Tsuchiya T, Kanegasaki S, et al. Aurantio-obtusin stimulates chemotactic migration and differentiation of MC3T3-E1 osteoblast cells. Planta Med, 2014, 80(7): 544-549.
14.	van der Eerden BC, Oei L, Roschger P, et al. TRPV4 deficiency causes sexual dimorphism in bone metabolism and osteoporotic fracture risk. Bone, 2013, 57(2): 443-454.
15.	Zhang Y, Xu J, Ruan YC, et al. Implant-derived magnesium induces local neuronal production of CGRP to improve bone-fracture healing in rats. Nat Med, 2016, 22(10): 1160-1169.
16.	Wachter RF, Briggs GP, Pedersen CE Jr. Precipitation of phase I antigen of Coxiella burnetii by sodium sulfite. Acta Virol, 1975, 19(6): 500.
17.	Bowman CL, Gottlieb PA, Suchyna TM, et al. Mechanosensitive ion channels and the peptide inhibitor GsMTx-4: history, properties, mechanisms and pharmacology. Toxicon, 2007, 49(2): 249-270.
18.	Bae C, Sachs F, Gottlieb PA. The mechanosensitive ion channel Piezo1 is inhibited by the peptide GsMTx4. Biochemistry, 2011, 50(29): 6295-6300.
19.	Wang S, Chennupati R, Kaur H, et al. Endothelial cation channel PIEZO1 controls blood pressure by mediating flow-induced ATP release. J Clin Invest, 2016, 126(12): 4527-4536.
20.	Evans EL, Cuthbertson K, Endesh N, et al. Yoda1 analogue (Dooku1) which antagonizes Yoda1-evoked activation of Piezo1 and aortic relaxation. Br J Pharmacol, 2018, 175(10): 1744-1759.
21.	Cahalan SM, Lukacs V, Ranade SS, et al. Piezo1 links mechanical forces to red blood cell volume. Elife, 2015, 4.
22.	Cheng G, Gao F, Sun X, et al. Paris saponin Ⅶ suppresses osteosarcoma cell migration and invasion by inhibiting MMP-2/9 production via the p38 MAPK signaling pathway. Mol Med Rep, 2016, 14(4): 3199-3205.
23.	焦雪峰, 黄永灿, 黄益洲, 等. 大鼠 BMSCs 自发钙化过程中成骨相关基因表达谱的分析. 中国修复重建外科杂志, 2014, 28(2): 133-141.
24.	Andolfi L, Bourkoula E, Migliorini E, et al. Investigation of adhesion and mechanical properties of human glioma cells by single cell force spectroscopy and atomic force microscopy. PLoS One, 2014, 9(11): e112582.

1. Sims NA, Martin TJ. Coupling the activities of bone formation and resorption: a multitude of signals within the basic multicellular unit. Bonekey Rep, 2014, 3: 481.
2. Seeman E. Bone modeling and remodeling. Crit Rev Eukaryot Gene Expr, 2009, 19(3): 219-233.
3. Lai CF, Cheng SL. Alphavbeta integrins play an essential role in BMP-2 induction of osteoblast differentiation. J Bone Miner Res, 2005, 20(2): 330-340.
4. 胥少汀, 葛宝丰, 徐印坎. 实用骨科学. 4 版. 北京: 人民军医出版社, 2012: 632-654.
5. Tang Y, Wu X, Lei W, et al. TGF-beta1-induced migration of bone mesenchymal stem cells couples bone resorption with formation. Nat Med, 2009, 15(7): 757-765.
6. Xian L, Wu X, Pang L, et al. Matrix IGF-1 maintains bone mass by activation of mTOR in mesenchymal stem cells. Nat Med, 2012, 18(7): 1095-1101.
7. Aryal A C S, Miyai K, Izu Y, et al. Nck influences preosteoblastic/osteoblastic migration and bone mass. Proc Natl Acad Sci U S A, 2015, 112(50): 15432-15437.
8. Coste B, Mathur J, Schmidt M, et al. Piezo1 and Piezo2 are essential components of distinct mechanically activated cation channels. Science, 2010, 330(6000): 55-60.
9. Sugimoto A, Miyazaki A, Kawarabayashi K, et al. Piezo type mechanosensitive ion channel component 1 functions as a regulator of the cell fate determination of mesenchymal stem cells. Sci Rep, 2017, 7(1): 17696.
10. Hansen TH, Madsen MTB, Jørgensen NR, et al. Bone turnover, calcium homeostasis, and vitamin D status in Danish vegans. Eur J Clin Nutr, 2018, 72(7): 1046-1054.
11. Campbell EJ, Campbell GM, Hanley DA. The effect of parathyroid hormone and teriparatide on fracture healing. Expert Opin Biol Ther, 2015, 15(1): 119-129.
12. Eleniste PP, Huang S, Wayakanon K, et al. Osteoblast differentiation and migration are regulated by dynamin GTPase activity. Int J Biochem Cell Biol, 2014, 46: 9-18.
13. Vishnuprasad CN, Tsuchiya T, Kanegasaki S, et al. Aurantio-obtusin stimulates chemotactic migration and differentiation of MC3T3-E1 osteoblast cells. Planta Med, 2014, 80(7): 544-549.
14. van der Eerden BC, Oei L, Roschger P, et al. TRPV4 deficiency causes sexual dimorphism in bone metabolism and osteoporotic fracture risk. Bone, 2013, 57(2): 443-454.
15. Zhang Y, Xu J, Ruan YC, et al. Implant-derived magnesium induces local neuronal production of CGRP to improve bone-fracture healing in rats. Nat Med, 2016, 22(10): 1160-1169.
16. Wachter RF, Briggs GP, Pedersen CE Jr. Precipitation of phase I antigen of Coxiella burnetii by sodium sulfite. Acta Virol, 1975, 19(6): 500.
17. Bowman CL, Gottlieb PA, Suchyna TM, et al. Mechanosensitive ion channels and the peptide inhibitor GsMTx-4: history, properties, mechanisms and pharmacology. Toxicon, 2007, 49(2): 249-270.
18. Bae C, Sachs F, Gottlieb PA. The mechanosensitive ion channel Piezo1 is inhibited by the peptide GsMTx4. Biochemistry, 2011, 50(29): 6295-6300.
19. Wang S, Chennupati R, Kaur H, et al. Endothelial cation channel PIEZO1 controls blood pressure by mediating flow-induced ATP release. J Clin Invest, 2016, 126(12): 4527-4536.
20. Evans EL, Cuthbertson K, Endesh N, et al. Yoda1 analogue (Dooku1) which antagonizes Yoda1-evoked activation of Piezo1 and aortic relaxation. Br J Pharmacol, 2018, 175(10): 1744-1759.
21. Cahalan SM, Lukacs V, Ranade SS, et al. Piezo1 links mechanical forces to red blood cell volume. Elife, 2015, 4.
22. Cheng G, Gao F, Sun X, et al. Paris saponin Ⅶ suppresses osteosarcoma cell migration and invasion by inhibiting MMP-2/9 production via the p38 MAPK signaling pathway. Mol Med Rep, 2016, 14(4): 3199-3205.
23. 焦雪峰, 黄永灿, 黄益洲, 等. 大鼠 BMSCs 自发钙化过程中成骨相关基因表达谱的分析. 中国修复重建外科杂志, 2014, 28(2): 133-141.
24. Andolfi L, Bourkoula E, Migliorini E, et al. Investigation of adhesion and mechanical properties of human glioma cells by single cell force spectroscopy and atomic force microscopy. PLoS One, 2014, 9(11): e112582.

Chinese Journal of Reparative and Reconstructive Surgery

Effect of knocking down Piezo1 mechanically sensitive protein on migration of MC3T3-E1 osteoblast cells

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