Effect of stretch on taurine upregulated gene 1-mediated miR-545-3p/cannbinoida receptor 2 pathway regulating distraction osteogenesis in rats_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

ZHANG Mengzhu ¹ ,  WANG Bin ² , WANG Zixin ¹ , WU Yalong ¹ , ZHENG Yongxin ¹

1. Department of Orthopedics, Affiliated Hospital of North China University of Science and Technology, Tangshan Hebei, 063000, P. R. China;
2. Comprehensive Office of Party and Government of Tangshan Second Hospital, Tangshan Hebei, 063000, P. R. China;

Corresponding author：

WANG Bin, Email: wbladyp3@163.com

Keywords：

Distraction osteogenesis; taurine upregulated gene 1; mineralization of distraction area; stretch; cannbinoida receptor 2

DOI：

10.7507/1002-1892.202503010

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Objective To investigate the effect of stretch on long non-coding RNA taurine upregulated gene 1 (TUG1)-mediated miR-545-3P/cannbinoida receptor 2 (CNR2) pathway regulating bone regeneration in the distraction area of rats during distraction osteogenesis. Methods Thirty-six 10-week-old male Sprague Dawley rats were randomly divided into 3 groups (n=12 in each group): group A (femoral fracture+injection of interfering RNA), group B (distraction osteogenesis+injection of interfering RNA), and group C (distraction osteogenesis+injection of TUG1). Groups A and B were injected with 60 μg of interfering RNA at the beginning of incubation period (immediate after operation), the beginning of distraction phase (7 days after operation), and the end of distraction phase (21 days after operation), and group C was injected with 60 μg of synthetic TUG1 in vivo interfering sequence at the same time. The general situation of rats in each group was observed during the experiment. The mineralization of fracture space or distraction area was observed by X-ray films on 21, 35, and 49 days after operation. At 49 days after operation, the samples of the distraction area were taken for HE staining to observe the mineralization, and real-time fluorescence quantitative PCR (qRT-PCR) was used to detect the expression of osteoblast-related genes such as TUG1, miR-545-3p, CNR2, alkaline phosphatase (ALP), osteocalcin (OCN), and osteopontin (OPN). Blood samples were collected from the abdominal aorta of the rats, and the expressions of ALP and C terminal telopeptide of type Ⅰ (CTX-Ⅰ) protein were detected by ELISA assay. Results The results of X-ray film and HE staining observations showed that osteogenesis in group C was more rapid than that in groups A and B at the same time point. The results of qRT-PCR showed that the relative mRNA expressions of TUG1, CNR2, ALP, OCN, and OPN in group C were significantly higher than those in group A and group B, and the relative mRNA expression of miR-545-3p in group C was significantly lower than that in group A and group B (P<0.05). The relative mRNA expressions of TUG1 and ALP in group B were significantly higher than those in group A, and the relative mRNA expression of miR-545-3p in group B was significantly lower than that in group A (P<0.05). There was no significant difference in the relative mRNA expressions of CNR2, OCN, and OPN between group A and group B (P>0.05). The results of ELISA showed that the expressions of ALP and CTX-Ⅰ protein in group C were significantly higher than those in group A and group B, and that in group B was significantly higher than that in group A (P<0.05). Conclusion Under the action of stretch force, the expression of TUG1 in the femoral extension area of rats increased, which promoted the expression of CNR2 by inhibiting the expression of miR-545-3P, which was helpful to the mineralization of the extension area and promoted osteogenesis.

1.	Silva AM, Moura SR, Teixeira JH, et al. Long noncoding RNAs: a missing link in osteoporosis. Bone Res, 2019, 7: 10. doi: 10.1038/s41413-019-0048-9.
2.	He Y, Chen Y. The potential role of lncRNAs in osteoporosis. J Bone Miner Metab, 2021, 39(3): 341-352.
3.	Zhou H, Sun L, Wan F. Molecular mechanisms of TUG1 in the proliferation, apoptosis, migration and invasion of cancer cells. Oncol Lett, 2019, 18(5): 4393-4402.
4.	Li W, Li L, Cui R, et al. Bone marrow mesenchymal stem cells derived exosomal Lnc TUG1 promotes bone fracture recovery via miR-22-5p/Anxa8 axis. Hum Cell, 2023, 36(3): 1041-1053.
5.	Yao Z, An W, Moming A, et al. Long non-coding RNA TUG1 knockdown repressed the viability, migration and differentiation of osteoblasts by sponging miR-214. Exp Ther Med, 2022, 23(3): 203. doi: 10.3892/etm.2022.11126.
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7.	王兴文. LncRNA TUG1/miR-34a/FGFR1轴在流体剪切应力调控成骨细胞生物学过程中的作用及机制研究. 兰州: 兰州大学, 2022.
8.	辜淑君, 陈德晖, 张抗抗, 等. 幼龄大鼠异位气管移植致闭塞性细支气管炎动物模型的构建. 中国病理生理杂志, 2021, 37(12): 2299-2304.
9.	Che J, Chen X, Ren W, et al. PTH 1-34 reduced apoptosis of MLO-Y4 osteocyte-like cells by activating autophagy and inhibiting ER stress under RPM conditions. Eur J Pharmacol, 2024, 967: 176364. doi: 10.1016/j.ejphar.2024.176364.
10.	Liu ES, Zalutskaya A, Chae BT, et al. Phosphate interacts with PTHrP to regulate endochondral bone formation. Endocrinology, 2014, 155(10): 3750-3756.
11.	Xie J, Bao M, Bruekers SMC, et al. Collagen gels with different fibrillar microarchitectures elicit different cellular responses. ACS Appl Mater Interfaces, 2017, 9(23): 19630-19637.
12.	Rim EY, Clevers H, Nusse R. The Wnt pathway: From signaling mechanisms to synthetic modulators. Annu Rev Biochem, 2022, 91: 571-598.
13.	Gao Y, Chen N, Fu Z, et al. Progress of Wnt signaling pathway in osteoporosis. Biomolecules, 2023, 13(3): 483. doi: 10.3390/biom13030483.
14.	Xie Y, Su N, Yang J, et al. FGF/FGFR signaling in health and disease. Signal Transduct Target Ther, 2020, 5(1): 181. doi: 10.1038/s41392-020-00222-7.
15.	Kamalitdinov TB, Fujino K, Keith LS, et al. Targeting the hedgehog signaling pathway to improve tendon-to-bone integration. Osteoarthritis Cartilage, 2023, 31(9): 1202-1213.
16.	Long Z, Dou P, Cai W, et al. MiR-181a-5p promotes osteogenesis by targeting BMP3. Aging (Albany NY), 2023, 15(3): 734-747.
17.	Akkurt MO, Demirkale I, Öznur A. Partial calcanectomy and Ilizarov external fixation may reduce amputation need in severe diabetic calcaneal ulcers. Diabet Foot Ankle, 2017, 8(1): 1264699. doi: 10.1080/2000625X.2017.1264699.
18.	杨琼琼, 李萍, 王倩. 牙种植体周围骨组织修复中microRNA的作用及调控机制. 中国组织工程研究, 2022, 26(28): 4539-4545.
19.	周雷, 王明海. 成骨分化相关信号通路的研究进展. 临床医学进展, 2017, 7(4): 235-241.
20.	张晓玲, 叶艳霞, 叶志华. miR-545-3p抑制VEGFA/VEGFR2信号通路对前列腺癌细胞增殖和凋亡的影响. 中国性科学, 2020, 29(9): 5-10.
21.	Liu SC, Sun QZ, Qiao XF, et al. LncRNA TUG1 influences osteoblast proliferation and differentiation through the Wnt/β-catenin signaling pathway. Eur Rev Med Pharmacol Sci, 2019, 23(11): 4584-4590.
22.	He Q, Yang S, Gu X, et al. Correction to: Long noncoding RNA TUG1 facilitates osteogenic differentiation of periodontal ligament stem cells via interacting with Lin28A. Cell Death Dis, 2018, 9(7): 710. doi: 10.1038/s41419-018-0750-3.
23.	赵子慧, 胡思婧, 陈高策, 等. 基于大麻素2型受体和雌激素受体α研究升麻和黑升麻含药血清对成骨细胞功能的影响. 中草药, 2023, 54(18): 5941-5951.
24.	胡思婧, 练晨霞, 张奇, 等. 熟地黄的大麻素2型受体激动剂活性及对骨代谢的调控作用研究. 中草药, 2022, 53(20): 6481-6491.
25.	吴文苑. 骨型碱性磷酸酶的检测方法和临床应用. 国外医学 (临床生物化学与检验学分册), 1999, 20(2): 64-65.
26.	Carvajal Alegria G, Garrigues F, Bettacchioli E, et al. Tocilizumab controls bone turnover in early polymyalgia rheumatica. Joint Bone Spine, 2021, 88(3): 105117. doi: 10.1016/j.jbspin.2020.105117.
27.	Chubb SA. Measurement of C-terminal telopeptide of type I collagen (CTX) in serum. Clin Biochem, 2012, 45(12): 928-935.
28.	Wei X, Wang J, Deng YY, et al. Tubiechong patching promotes tibia fracture healing in rats by regulating angiogenesis through the VEGF/ERK1/2 signaling pathway. J Ethnopharmacol, 2023, 301: 115851. doi: 10.1016/j.jep.2022.115851.

1. Silva AM, Moura SR, Teixeira JH, et al. Long noncoding RNAs: a missing link in osteoporosis. Bone Res, 2019, 7: 10. doi: 10.1038/s41413-019-0048-9.
2. He Y, Chen Y. The potential role of lncRNAs in osteoporosis. J Bone Miner Metab, 2021, 39(3): 341-352.
3. Zhou H, Sun L, Wan F. Molecular mechanisms of TUG1 in the proliferation, apoptosis, migration and invasion of cancer cells. Oncol Lett, 2019, 18(5): 4393-4402.
4. Li W, Li L, Cui R, et al. Bone marrow mesenchymal stem cells derived exosomal Lnc TUG1 promotes bone fracture recovery via miR-22-5p/Anxa8 axis. Hum Cell, 2023, 36(3): 1041-1053.
5. Yao Z, An W, Moming A, et al. Long non-coding RNA TUG1 knockdown repressed the viability, migration and differentiation of osteoblasts by sponging miR-214. Exp Ther Med, 2022, 23(3): 203. doi: 10.3892/etm.2022.11126.
6. Hao R, Wang B, Wang H, et al. lncRNA TUG1 promotes proliferation and differentiation of osteoblasts by regulating the miR-545-3p/CNR2 axis. Braz J Med Biol Res, 2020, 53(11): e9798. doi: 10.1590/1414-431X20209798.
7. 王兴文. LncRNA TUG1/miR-34a/FGFR1轴在流体剪切应力调控成骨细胞生物学过程中的作用及机制研究. 兰州: 兰州大学, 2022.
8. 辜淑君, 陈德晖, 张抗抗, 等. 幼龄大鼠异位气管移植致闭塞性细支气管炎动物模型的构建. 中国病理生理杂志, 2021, 37(12): 2299-2304.
9. Che J, Chen X, Ren W, et al. PTH 1-34 reduced apoptosis of MLO-Y4 osteocyte-like cells by activating autophagy and inhibiting ER stress under RPM conditions. Eur J Pharmacol, 2024, 967: 176364. doi: 10.1016/j.ejphar.2024.176364.
10. Liu ES, Zalutskaya A, Chae BT, et al. Phosphate interacts with PTHrP to regulate endochondral bone formation. Endocrinology, 2014, 155(10): 3750-3756.
11. Xie J, Bao M, Bruekers SMC, et al. Collagen gels with different fibrillar microarchitectures elicit different cellular responses. ACS Appl Mater Interfaces, 2017, 9(23): 19630-19637.
12. Rim EY, Clevers H, Nusse R. The Wnt pathway: From signaling mechanisms to synthetic modulators. Annu Rev Biochem, 2022, 91: 571-598.
13. Gao Y, Chen N, Fu Z, et al. Progress of Wnt signaling pathway in osteoporosis. Biomolecules, 2023, 13(3): 483. doi: 10.3390/biom13030483.
14. Xie Y, Su N, Yang J, et al. FGF/FGFR signaling in health and disease. Signal Transduct Target Ther, 2020, 5(1): 181. doi: 10.1038/s41392-020-00222-7.
15. Kamalitdinov TB, Fujino K, Keith LS, et al. Targeting the hedgehog signaling pathway to improve tendon-to-bone integration. Osteoarthritis Cartilage, 2023, 31(9): 1202-1213.
16. Long Z, Dou P, Cai W, et al. MiR-181a-5p promotes osteogenesis by targeting BMP3. Aging (Albany NY), 2023, 15(3): 734-747.
17. Akkurt MO, Demirkale I, Öznur A. Partial calcanectomy and Ilizarov external fixation may reduce amputation need in severe diabetic calcaneal ulcers. Diabet Foot Ankle, 2017, 8(1): 1264699. doi: 10.1080/2000625X.2017.1264699.
18. 杨琼琼, 李萍, 王倩. 牙种植体周围骨组织修复中microRNA的作用及调控机制. 中国组织工程研究, 2022, 26(28): 4539-4545.
19. 周雷, 王明海. 成骨分化相关信号通路的研究进展. 临床医学进展, 2017, 7(4): 235-241.
20. 张晓玲, 叶艳霞, 叶志华. miR-545-3p抑制VEGFA/VEGFR2信号通路对前列腺癌细胞增殖和凋亡的影响. 中国性科学, 2020, 29(9): 5-10.
21. Liu SC, Sun QZ, Qiao XF, et al. LncRNA TUG1 influences osteoblast proliferation and differentiation through the Wnt/β-catenin signaling pathway. Eur Rev Med Pharmacol Sci, 2019, 23(11): 4584-4590.
22. He Q, Yang S, Gu X, et al. Correction to: Long noncoding RNA TUG1 facilitates osteogenic differentiation of periodontal ligament stem cells via interacting with Lin28A. Cell Death Dis, 2018, 9(7): 710. doi: 10.1038/s41419-018-0750-3.
23. 赵子慧, 胡思婧, 陈高策, 等. 基于大麻素2型受体和雌激素受体α研究升麻和黑升麻含药血清对成骨细胞功能的影响. 中草药, 2023, 54(18): 5941-5951.
24. 胡思婧, 练晨霞, 张奇, 等. 熟地黄的大麻素2型受体激动剂活性及对骨代谢的调控作用研究. 中草药, 2022, 53(20): 6481-6491.
25. 吴文苑. 骨型碱性磷酸酶的检测方法和临床应用. 国外医学 (临床生物化学与检验学分册), 1999, 20(2): 64-65.
26. Carvajal Alegria G, Garrigues F, Bettacchioli E, et al. Tocilizumab controls bone turnover in early polymyalgia rheumatica. Joint Bone Spine, 2021, 88(3): 105117. doi: 10.1016/j.jbspin.2020.105117.
27. Chubb SA. Measurement of C-terminal telopeptide of type I collagen (CTX) in serum. Clin Biochem, 2012, 45(12): 928-935.
28. Wei X, Wang J, Deng YY, et al. Tubiechong patching promotes tibia fracture healing in rats by regulating angiogenesis through the VEGF/ERK1/2 signaling pathway. J Ethnopharmacol, 2023, 301: 115851. doi: 10.1016/j.jep.2022.115851.

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Latest ArticlesEffect of stretch on taurine upregulated gene 1-mediated miR-545-3p/cannbinoida receptor 2 pathway regulating distraction osteogenesis in rats

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