miR-206靶向Cx43调控ERK1/2通路在兔激素性股骨头坏死中的作用

doi:10.13418/j.issn.1001-165x.2021.02.008

PDF(6412 KB)

中国临床解剖学杂志 ›› 2021, Vol. 39 ›› Issue (2) : 154-160. DOI: 10.13418/j.issn.1001-165x.2021.02.008

实验研究

miR-206靶向Cx43调控ERK1/2通路在兔激素性股骨头坏死中的作用

席源^{1, 2}，　罗高斌¹，　魏桂清¹，　覃文涛¹，　薄占东¹

作者信息 +

The role of miR-206 on regulation of ERK1/2 signaling pathway by targeting Cx43 in rabbit steroid-induced avascular necrosis of femoral head model

Xi Yuan^1,2, Luo Gaobin¹，Wei Guiqing¹，Qin Wentao¹, Bo Zhandong¹

Author information +

文章历史 +

摘要

目的　探讨微小RNA（MicroRNA，miR）-206调控缝隙连接蛋白（connexin，Cx）43激活蛋白激酶（ERK）1/2通路是否参与兔激素性股骨头坏死的发生发展，并初步阐明其机制。方法　成年新西兰大白兔60只，随机分为实验组和对照组各30只，实验组接受内毒素（10 mg/kg）联合甲强龙（20 mg/kg）注射制作激素性股骨头坏死模型。注射2周、8周和16周后两组动物行MRI检查，HE染色确定模型建立成功；模型兔与相应对照组股骨头标本行原位杂交和实时荧光定量PCR（qPCR）检测miR-206表达，Western blot、免疫组织化学和qPCR检测Cx43、ERK1/2和Runx2基因及蛋白表达。结果　建模成功率为70%；原位杂交显示miR-206表达定位于兔股骨头髓腔、成骨细胞及少量骨细胞。造模后2周、8周和16周，与对照组比较，实验组兔股骨头内miR-206表达上调；Cx43、Runx2基因表达下调，Cx43、ERK1/2、Runx2蛋白表达下调。结论　miR-206可通过下调其靶蛋白Cx43，抑制ERK1/2信号通路，抑制成骨分化，参与激素性股骨头坏死发生发展及修复。

Abstract

Objective　To explore the role of MicroRNA(miR)-206 on regulating of extracellular signal-regulated protein kinase(ERK) 1/2 signaling pathway by targeting connexin(Cx) 43 in rabbit model of steroid-induced avascular necrosis of femoral head.　Methods　Sixty mature rabbits were randomly divided into a model group (n=30) and a control group (n=30). Rabbit model of femoral head necrosis was made with lipopolysaccharide (LPS) and methylprednisolone (MPS). The establishment of the model was determined by MRI and HE staining after modeled 2、8 and 16 weeks. Total RNA and protein were extracted from the femoral head. In situ hybridization and Quantitative Real-time PCR (qPCR) were employed to detect the change of miRNA-206; qPCR, western blot and immunohistochemistry were used to detect the expression of Cx43、ERK1/2 and Runx2.　Results　The model success rate was 70%. In situ hybridization result showed that the miR-206 expressed in rabbit femoral head medullary space, osteoblasts and osteocyte. Compared with the control group, miR-206 expression of the model group up-regulated at 2 weeks, 8 weeks and 16 weeks after modeling, the expression of Cx43 and Runx2 mRNA in the model group down-regulated, the expression of Cx43、ERK1/2 and Runx2 protein in the model group also down-regulated.　Conclusions miR-206 can be involved in the occurrence, development and repair of steroid-induced femoral head necrosis by down-regulating its target protein CX43, inhibiting the ERK1/2 signaling pathway and osteogenic differentiation.

导出引用

席源, 罗高斌, 魏桂清, 覃文涛, 薄占东. miR-206靶向Cx43调控ERK1/2通路在兔激素性股骨头坏死中的作用[J]. 中国临床解剖学杂志. 2021, 39(2): 154-160 https://doi.org/10.13418/j.issn.1001-165x.2021.02.008

Xi Yuan, Luo Gaobin, Wei Guiqing, Qin Wentao, Bo Zhandong. The role of miR-206 on regulation of ERK1/2 signaling pathway by targeting Cx43 in rabbit steroid-induced avascular necrosis of femoral head model[J]. Chinese Journal of Clinical Anatomy. 2021, 39(2): 154-160 https://doi.org/10.13418/j.issn.1001-165x.2021.02.008

中图分类号： R681.8

参考文献

[1] Wang XS, Zhuang QY, Weng XS, et al. Etiological and clinical analysis of osteonecrosis of the femoral head in Chinese patients[J]. Chin Med J (Engl), 2013, 126(2): 290-295. PMID: 23324279.
[2] Al-Omari AA, Aleshawi AJ, Marei OA, et al. Avascular necrosis of the femoral head after single steroid intra-articular injection[J]. Eur J Orthop Surg Traumatol, 2020, 30(2): 193-197. DOI: 10.1007/s00590-019-02555-8.
[3] Lai SW, Lin CL, Liao KF. Evaluating the association between avascular necrosis of femoral head and oral corticosteroids use in Taiwan[J]. Medicine (Baltimore), 2020, 99(3): e18585. DOI: 10.1097/MD. 00000 00000018585.
[4] Liu G, Luo GB, Bo ZD, et al. Impaired osteogenic differentiation associated with connexin 43/microRNA-206 in steroid-induced avascular necrosis of the femoral head[J]. Exp Mol Pathol, 2016, 101(1): 89-99. DOI: 10.1016/j.yexmp.2016.07.009.
[5] Niger C, Buo AM, Hebert C, et al. ERK acts in parallel to PKCdelta to mediate the connexin43-dependent potentiation of Runx2 activity by FGF2 in MC3T3 osteoblasts[J]. Am J Physiol Cell Physiol, 2012, 302(7): C1035-C1044. DOI: 10.1152/ajpcell.00262.2011.
[6] Weinstein RS, Jilka RL, Parfitt AM, et al. Inhibition of osteoblastogenesis and promotion of apoptosis of osteoblasts and osteocytes by glucocorticoids. Potential mechanisms of their deleterious effects on bone[J]. J Clin Invest, 1998, 102(2): 274-282. DOI: 10.1172/JCI2799.
[7] 康鹏德, 裴福兴, 杨静, 等. 糖皮质激素诱导骨细胞脂肪化与股骨头坏死的病理机制研究[J]. 中华骨科杂志, 2013, 33(7): 762-769. DOI: 10.3760/cma.j.issn.0253-2352.2013.07.013.
[8] Kim J, Ko J. A novel PPAR gamma2 modulator sLZIP controls the balance between adipogenesis and osteogenesis during mesenchymal stem cell differentiation[J]. Cell Death Differ, 2014, 21(10): 1642-1655. DOI: 10.1038/cdd.2014.80.
[9] Hernigou P. Cellular therapy for the treatment of osteonecrosis: from bench to bedside[J]. Instr Course Lect, 2020, 69: 139-148. PMID: 32017725.
[10]马信龙, 刘泽朋, 马剑雄, 等. 激紊性股骨头坏死股骨头内Runx2、Osterix及AJ18的动态表达[J]. 中华骨科杂志, 2010, 30(1): 67-72. DOI: 10.3760/cma.j.issn.0253-2352.2010.01.016.
[11]Ciesla M, Skrzypek K, Kozakowska M, et al. MicroRNAs as biomarkers of disease onset[J]. Anal Bioanal Chem, 2011, 401(7): 2051-2061. DOI: 10.1007/s00216-011-5001-8.
[12] McCarthy JJ. MicroRNA-206: the skeletal muscle-specific myomiR[J]. Biochim Biophys Acta, 2008, 1779(11): 682-691. DOI: 10.1016/j.bbagrm.2008.03.001.
[13] Inose H, Ochi H, Kimura A, et al. A microRNA regulatory mechanism of osteoblast differentiation[J]. Proc Natl Acad Sci U S A, 2009, 106(49): 20794-20799. DOI: 10.1073/pnas.0909311106.
[14] Zhang ZH, Jin AM, Yan DL. MicroRNA206 contributes to the progression of steroidinduced avascular necrosis of the femoral head by inducing osteoblast apoptosis by suppressing programmed cell death 4[J]. Mol Med Rep, 2018, 17(1): 801-808. DOI: 10.3892/mmr.2017.7963.
[15]Chen Y, Yang YR, Fan XL, et al. miR-206 inhibits osteogenic differentiation of bone marrow mesenchymal stem cells by targetting glutaminase[J]. Biosci Rep, 2019, 39(3): R20181108. DOI: 10.1042/BSR20181108.
[16]Anderson C, Catoe H, Werner R. MIR-206 regulates connexin43 expression during skeletal muscle development[J]. Nucleic Acids Res, 2006, 34(20): 5863-5871. DOI: 10.1093/nar/gkl743.
[17]Lima F, Niger C, Hebert C, et al. Connexin43 potentiates osteoblast responsiveness to fibroblast growth factor 2 via a protein kinase C-delta/Runx2-dependent mechanism[J]. Mol Biol Cell, 2009, 20(11): 2697-2708. DOI: 10.1091/mbc.e08-10-1079.
[18]Lin FX, Zheng GZ, Chang B, et al. Connexin 43 modulates osteogenic differentiation of bone marrow stromal cells through GSK-3beta/beta-catenin signaling pathways[J]. Cell Physiol Biochem, 2018, 47(1): 161-175. DOI: 10.1159/000489763.
[19] Abdallah BM, Ali EM. Butein promotes lineage commitment of bone marrow-derived stem cells into osteoblasts via modulating ERK1/2 signaling pathways[J]. Molecules, 2020, 25(8): 1885. DOI: 10.3390/molecules25081885.
[20] Lee JS, Kim ME, Seon JK, et al. Bone-forming peptide-3 induces osteogenic differentiation of bone marrow stromal cells via regulation of the ERK1/2 and Smad1/5/8 pathways[J]. Stem Cell Res, 2018, 26: 28-35. DOI: 10.1016/j.scr.2017.11.016.
[21] Lee CH, Huang YL, Liao JF, et al. Ugonin K promotes osteoblastic differentiation and mineralization by activation of p38 MAPK- and ERK-mediated expression of Runx2 and osterix[J]. Eur J Pharmacol, 2011, 668(3): 383-389. DOI: 10.1016/j.ejphar.2011.06.059.
[22]Gupta A, Leser JM, Gould NR, et al. Connexin43 regulates osteoprotegerin expression via ERK1/2 -dependent recruitment of Sp1[J]. Biochem Biophys Res Commun, 2019, 509(3): 728-733. DOI: 10.1016/j.bbrc.2018.12.173.