Effect of miR-199a regulating IGF1 expression on differentiation of osteoblasts under mechanical stimulation

doi:10.19439/j.sjos.2022.02.004

Abstract

Abstract: PURPOSE: To investigate the expression change of microRNA (miR) - 199a in MC3T3-E1 cells stimulated by mechanical stretch and its mechanism of osteogenic differentiation. METHODS: MC3T3-E1 cells cultured in vitro were loaded with 12% stretch for 0, 3, 6, 12 and 24 hours, alkaline phosphatase (ALP) activity was detected by ALP activity test kit, the expressions of osteocalcin (OCN), osteoblast specific transcription factor osterix (OSX), Runt related transcription factor 2 (Runx2) mRNA and miR-199a were detected by real-time fluorescence quantitative PCR. MC3T3-E1 cells were divided into control group, stretch group, stretch + miR-NC group and stretch + miR-199a group, and the expressions of miR-199a, OCN, OSX, Runx2 mRNA and protein and ALP activity were observed after 12% stretch and transfection of miR-199a. Alizarin red S (ARS) staining was used to observe calcium nodule formation ability. The target relationship between miR-199a and insulin like growth factor-1 (IGF1) was detected by double luciferase reporter gene assay; in addition, the effect of miR-199a mimic on IGF1 mRNA and protein expression was detected by real-time fluorescent quantitative PCR and Western blot. SPSS 24.0 software package was used to analyze the data. RESULTS: Compared with those at the time point of 0 h, ALP activity and expression level of OCN, OSX and Runx2 mRNA of MC3T3-E1 cells at 3, 6, 12 and 24 hours after mechanical stretch stimulation were significantly higher, while the expression level of miR-199a was significantly lower(P<0.05), and the change was most significant at 12 h. Compared with those in the control group, the expression level of miR-199a was significantly lower in the stretch group, while ALP activity, the expression level of OCN, OSX and Runx2 mRNA and protein, calcium nodule formation level were significantly high in the stretch group(P<0.05); there was no significant difference in the above indexes between the stretch group and stretch + miR-NC group(P>0.05). Compared with stretch + miR-NC group, the expression level of miR-199a in stretch + miR-199a group was significantly higher; while ALP activity, OCN, OSX, Runx2 mRNA and protein expression level, calcium nodule formation level were significantly lower(P<0.05). miR-199a could targetedly bind to IGF1, and the expression level of IGF1 mRNA and protein in MC3T3-E1 cells was significantly reduced by miR-199a mimic(P<0.05). CONCLUSIONS: MiR-199a can inhibit the osteogenic differentiation of MC3T3-E1 cells induced by mechanical stretch stimulation, and its mechanism may be related to the targeted regulation of IGF1 expression.

Key words: Osteogenic differentiation, Mechanical stimulation, MicroRNA-199a, Insulin like growth factor-1

CLC Number:

Q274

LIN Wei-long, WU Xiao-pei, WANG Xiao-ming, HE Wei-wei. Effect of miR-199a regulating IGF1 expression on differentiation of osteoblasts under mechanical stimulation[J]. Shanghai Journal of Stomatology, 2022, 31(2): 132-137.

References

[1] Haggard CA, Pumphrey BJ, Richman CS, et al.Enhancing periodontal regenerative outcomes with simultaneous orthodontic tooth movement[J]. Compend Contin Educ Dent, 2019, 40(1): 36-44.
[2] Huang H, Williams RC, Kyrkanides S.Accelerated orthodontic tooth movement: molecular mechanisms[J]. Am J Orthod Dentofacial Orthop, 2014, 146(5):620-632.
[3] Cui Q, Xing J, Yu M, et al.Mmu-miR-185 depletion promotes osteogenic differentiation and suppresses bone loss in osteoporosis through the Bgn-mediated BMP/Smad pathway[J]. Cell Death Dis, 2019, 10(3): 172-186.
[4] Tao GL, Mao P, Guan HN, et al.Effect of miR-181a-3p on osteogenic differentiation of human bone marrow-derived mesenchymal stem cells by targeting BMP10[J]. Artif Cells Nanomed Biotechnol, 2019, 47(1): 4159-4164.
[5] Wang T, Zhang C, Wu C, et al.miR-765 inhibits the osteogenic differentiation of human bone marrow mesenchymal stem cells by targeting BMP6 via regulating the BMP6/Smad1/5/9 signaling pathway[J]. Stem Cell Res Ther, 2020, 11(1): 62-73.
[6] Laine SK, Alm JJ, Virtanen SP, et al.MicroRNAs miR-96, miR-124, and miR-199a regulate gene expression in human bone marrow-derived mesenchymal stem cells[J]. J Cell Biochem, 2012, 113(8): 2687-2695.
[7] Wu JC, Sun J, Xu JC, et al.Down-regulated microRNA-199a-3p enhances osteogenic differentiation of bone marrow mesenchymal stem cells by targeting Kdm3a in ovariectomized rats[J]. Biochem J, 2021, 478(4): 721-734.
[8] 王高尚, 詹学斌, 赵育洁. miRNAs在心血管疾病中的研究进展[J]. 中国综合临床, 2020, 36(1): 92-96.
[9] Hu R, Zhu X, Chen C, et al.RNA-binding protein PUM2 suppresses osteosarcoma progression via partly and competitively binding to STARD13 3'UTR with miRNAs[J]. Cell Prolif, 2018, 51(6): 2508-2518.
[10] 孙芬, 刘铭, 刘名燕. 牵引力介导MC3T3-E1细胞microRNA的差异表达[J]. 口腔医学, 2018, 38(3): 222-226.
[11] Zeng Q, Wang Y, Gao J, et al.miR-29b-3p regulated osteoblast differentiation via regulating igf-1 secretion of mechanically stimulated osteocytes[J]. Cell Mol Biol Lett, 2019, 24: 11-23.
[12] Liu L, Liu M, Li R, et al.MicroRNA-503-5p inhibits stretch-induced osteogenic differentiation and bone formation[J]. Cell Biol Int, 2017, 41(2):112-123.
[13] 孙芬, 刘名燕, 刘铭, 等. miR-132-3p过表达对MC3T3-E1细胞增殖分化的影响[J]. 中国组织工程研究, 2020, 24(1): 59-64.
[14] 胡文佳, 陈晨, 黄辉, 等. miR-199a家族与肿瘤关系的研究进展[J]. 医学综述, 2019, 25(2): 280-285.
[15] Zeng B, Shi W, Tan G.MiR-199a/b-3p inhibits gastric cancer cell proliferation via down-regulating PAK4/MEK/ERK signaling pathway[J]. BMC Cancer, 2018, 18(1):34-40.
[16] 周懿, 郑浩, 杨远, 等. miR-199a通过靶向CTGF抑制结直肠癌细胞增殖和迁移[J]. 中国医药生物技术, 2019, 14(2): 142-148.
[17] 冬梅, 武剑, 韩杏梅, 等. 绝经后骨质疏松症患者胰岛素样生长因子1与相关骨代谢指标的关系[J]. 中国组织工程研究, 2017, 21(36):5862-5867.
[18] Littlejohn EL, Scott D, Saatman KE, et al.Insulin-like growth factor-1 overexpression increases long-term survival of posttrauma-born hippocampal neurons while inhibiting ectopic migration following traumatic brain injury[J]. Acta Neuropathol Commun, 2020, 8(1): 46-60.
[19] 邹晓岚, 叶茸茸, 王洋. 胰岛素样生长因子1及其受体在肿瘤中的研究进展[J]. 重庆医学, 2018, 47(18): 2474-2476, 2479.
[20] 易知非, 周芳, 谢增如. 胰岛素样生长因子1(IGF-1)通过抑制RUNX2促进骨折愈合的机制研究[J]. 医学研究杂志, 2018, 47(10): 27-31.
[21] Guo Y, Tang CY, Man XF, et al.Insulin-like growth factor-1 promotes osteogenic differentiation and collagen I alpha 2 synthesis via induction of mRNA-binding protein LARP6 expression[J]. Dev Growth Differ, 2017, 59(2): 94-103.