国际口腔医学杂志 ›› 2019, Vol. 46 ›› Issue (4): 420-425.doi: 10.7518/gjkq.2019048

• 论著 • 上一篇    下一篇

CaMKⅡδ基因沉默对破骨细胞分化功能及c-fos/c-jun/CREB基因的影响

张誉泓1,戚孟春1(),董伟1,孙红2   

  1. 1. 华北理工大学口腔医学院口腔颌面外科教研室 唐山 063000
    2. 华北理工大学基础医学院病理教研室 唐山 063000
  • 收稿日期:2018-07-22 修回日期:2019-03-01 出版日期:2019-07-10 发布日期:2019-07-12
  • 作者简介:张誉泓,医师,硕士,Email: 1293107508@qq.com
  • 基金资助:
    国家自然科学基金(81270965);河北省自然科学基金(20-17209114)

Effect of CaMKⅡδ gene silencing on osteoclast differentiation and c-fos/c-jun/CREB gene

Zhang Yuhong1,Qi Mengchun1(),Dong Wei1,Sun Hong2   

  1. 1. Dept. of Oral and Maxillofacial Surgery, College of Stomatology, North China University of Science and Technology, Tangshan 063000, China
    2. Dept. of Pathology, College of Basic Medicine, North China University of Science and Technology, Tangshan 063000, China
  • Received:2018-07-22 Revised:2019-03-01 Online:2019-07-10 Published:2019-07-12
  • Supported by:
    This study was supported by National Natural Science Foundation of China(81270965);National Natural Science Foundation of Hebei Province(20-17209114)

摘要:

目的 探索编码钙调蛋白依赖性激酶(CaMK)Ⅱδ的基因沉默对破骨细胞分化功能的影响以及c-fosc-junCREB基因在CaMKⅡδ调控破骨细胞分化中的作用,以揭示破骨细胞分化调控的分子机制。方法 构建CaMKⅡδ RNA干扰载体,转染至小鼠RAW264.7细胞,确定干扰效率。病毒转染细胞后,用50 ng·mL -1核因子-κB受体活化因子配体(RANKL)诱导,5 d后收获,通过耐酒石酸酸性磷酸酶(TRAP)检测、牛骨吸收陷窝检测沉默CaMKⅡδ基因对破骨细胞分化、骨吸收功能的影响;48 h后收获,利用实时聚合酶链式反应(PCR)、Western-blot等方法检测c-fosc-junCREB的表达情况。 结果 CaMKⅡδ RNA干扰在mRNA和蛋白质水平的干扰效率分别为70.6%和72.2%。 CaMKⅡδ RNA干扰可显著降低 c-fosc-junCREB的mRNA水平,下调幅度分别为74%、49% 和24%,CaMKⅡδ RNA干扰可显著降低c-Fos、c-Jun和CREB的水平,下调幅度分别为74.3%、61.3%和59.2%。结论 CaMKⅡδ RNA干扰可在mRNA和蛋白质水平显著抑制c-fosc-junCREB的表达,其共同参与CaMKⅡδ对破骨细胞分化的调控。

关键词: 破骨细胞, 钙调蛋白依赖性激酶Ⅱδ, c-Fos, c-Jun, 环腺苷酸反应原件结合蛋白

Abstract:

Objective This study aims to explore the effect of calmodulin-dependent kinase (CaMK) Ⅱδ gene silencing on osteoclast differentiation, investigate the roles of the c-fos, c-jun and CREB genes in CaMKⅡδ regulation of osteoclast differentiation, and reveal the molecular mechanism underlying osteoclast differentiation.Methods A CaMKⅡδ RNA interference vector was constructed and transfected into mouse RAW264.7 cells to determine interference efficiency. The cells were transfected with the virus, induced with 50 ng·mL -1 nuclear factor-κB receptor activator ligand and then harvested after 5 days. The effect of silencing the CaMKⅡδ gene on osteoclast differentiation was detected by tartrate-resistant acid phosphatase detection and bovine bone resorption lacuna. After 48 h, the expression levels of the c-fos, c-jun and CREB genes were detected by real-time polymerase chain reaction and Western blot.Results The interference efficiencies of the CaMKⅡδ RNA interference vector at the mRNA and protein levels were 70.6% and 72.2%, respectively. CaMKⅡδ RNA interference significantly reduced the mRNA levels of c-fos, c-jun and CREB by 74%, 49% and 24%, respectively. CaMKⅡδ RNA interference significantly reduced the protein levels of c-Fos, c-Jun and CREB by 74.3%, 61.3% and 59.2%, respectively.Conclusion CaMKⅡδ RNA interference can significantly inhibit the expression of c-fos, c-jun and CREB at the mRNA and protein levels. These results indicate that c-Fos, c-Jun and CREB are involved in the regulatory effects of CaMKⅡδ on osteoclast differentiation.

Key words: osteoclast, calmodulin-dependent kinase Ⅱδ, c-Fos, c-Jun, cyclic adenosine monophosphate responsive element-binding protein

中图分类号: 

  • Q786

图 1

实时PCR检测CaMKⅡδ mRNA水平 ▼与对照组比较,P>0.05;●与对照组及阴性载体组比较,P<0.01。n=3。"

图 2

Western-blotting检测CaMKⅡδ表达水平"

图 3

TRAP、牛骨吸收陷窝检测破骨细胞的形成 A、D:对照组;B、E:阴性载体组;C、F:干扰载体组。A~C:TRAP染色;D~F:SEM。"

图 4

实时PCR检测c-fos mRNA、c-jun mRNA和CREB mRNA水平 阴性载体组与对照组比较,P>0.05;**干扰载体组c-fos和CREB mRNA水平与对照组及阴性载体组比较,P<0.01;*干扰载体组c-jun mRNA水平与对照组及阴性载体组比较,P<0.05。n=3。"

图 5

Western-blotting检测c-Fos、c-Jun和CREB表达"

[1] Pereira M, Petretto E, Gordon S , et alCommon signalling pathways in macrophage and osteoclast multinucleation[J]. J Cell Sci, 2018,131:11. pii:jcs216267.
[2] Yuan FL, Wu QY, Miao ZN , et al. Osteoclast-derived extracellular vesicles: novel regulators of osteoclastogenesis and osteoclast-osteoblasts communication in bone remodeling[J]. Front Physiol, 2018,9:628.
[3] Yao CH, Zhang P, Zhang L . Differential protein and mRNA expression of CaMKs during osteoclastogenesis and its functional implications[J]. Biochem Cell Biol, 2012,90(4):532-539.
[4] Kim JY, Cheon YH, Yoon KH , et al. Parthenolide inhibits osteoclast differentiation and bone resorbing activity by down-regulation of NFATc1 induction and c-Fos stability, during RANKL-mediated osteoclastogenesis[J]. BMB Rep, 2014,47(8):451-456.
[5] Mohamed SG, Sugiyama E, Shinoda K , et al. Interleukin-10 inhibits RANKL-mediated expression of NFATc1 in part via suppression of c-Fos and c-Jun in RAW264.7 cells and mouse bone marrow cells[J]. Bone, 2007,41(4):592-602.
doi: 10.1016/j.bone.2007.05.016
[6] Park KH, Gu DR, Jin SH , et al. Pueraria lobate inhibits RANKL-mediated osteoclastogenesis via dow-nregulation of CREB/PGC1β/c-Fos/NFATc1 signaling[J]. Am J Chin Med, 2017,45(8):1725-1744.
[7] 刘娟娟, 董伟, 戚孟春 , 等. 钙离子/钙调蛋白依赖性蛋白激酶Ⅱδ RNA干扰对下游基因表达及破骨细胞分化的影响[J]. 第三军医大学学报, 2017,39(1):22-27.
Liu JJ, Dong W, Qi MC , et al. Influence of CaMK Ⅱ δ RNA interference on expression of its downstream molecules and osteoclast differentiation[J]. J Third Mil Med Univ, 2017,39(1):22-27.
[8] Teitelbaum SL . Bone resorption by osteoclasts[J]. Science, 2000,289(5484):1504-1508.
[9] Matsuo K, Galson DL, Zhao C , et al. Nuclear factor of activated T-cells (NFAT) rescues osteoclastogenesis in precursors lacking c-Fos[J]. J Biol Chem, 2004,279(25):26475-26480.
[10] Matsuo K, Ray N . Osteoclasts, mononuclear phagocytes, and c-Fos: new insight into osteoimmunology[J]. Keio J Med, 2004,53(2):78-84.
[11] Nie S, Xu J, Zhang C , et al. Salicortin inhibits osteoclast differentiation and bone resorption by down-regulating JNK and NF-κB/NFATc1 signaling pathways[J]. Biochem Biophys Res Commun, 2016,470(1):61-67.
[12] 王想福, 孙凤歧, 石瑞芳 , 等. 靶向抑制PI3K对人体外周血破骨细胞分化p38/c-Fos信号通路调控的研究[J]. 中国骨质疏松杂志, 2016,22(1):49-52.
Wang XF, Sun FQ, Shi RF , et al. The regulation of p38/c-Fos signal pathway by targeting inhibition of PI3K in human peripheral osteoclasts[J]. Chin J Osteopor, 2016,22(1):49-52.
[13] Izawa T, Arakaki R, Mori H , et al. The nuclear receptor AhR controls bone homeostasis by regulating osteoclast differentiation via the RANK/c-Fos signaling Axis[J]. J Immunol, 2016,197(12):4639-4650.
[14] 王红美, 董伟, 戚孟春 , 等. CREB和ERK1/2在CaMKⅡδ调控破骨细胞分化中的作用[J]. 解放军医学杂志, 2018,43(2):91-95.
Wang HM, Dong W, Qi MC , et al. Regulation of osteoclast differentiation by CaMKⅡδ mediated through CREB and ERK1/2 signal pathways[J]. Med J Chin PLA, 2018,43(2):91-95.
[15] 陆大壮, 刘娟娟, 戚孟春 , 等. CaMKⅡδ在破骨细胞分化不同阶段表达规律的研究[J]. 中国病理生理杂志, 2016,32(10):1870-1874.
Lu DZ, Liu JJ, Qi MC , et al. Expression profiles of CaMKⅡδ at different stages of osteoclast differen-tiation[J]. Chin J Pathophysiol, 2016,32(10):1870-1874.
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