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
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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-fos、c-jun、CREB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CaMKⅡδRNA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)、Westernblot等方法检测c-fos、c-jun、CREB的表达情况。结果 CaMKⅡδRNA干扰在mRNA和蛋白质水平的干扰效率分别为70.6%和72.2%。CaMKⅡδRNA干扰可显著降低c-fos、c-jun和CREB的mRNA水平,下调幅度分别为74%、49%和24%,CaMKⅡδRNA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 CaMKⅡδRNA干扰可在mRNA和蛋白质水平显著抑制c-fos、c-jun、CREB的表达,其共同参与CaMKⅡδ对破骨细胞分化的调控。
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 tartrateresistant 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 by74%, 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.
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 tartrateresistant 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 by74%, 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.
引文
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[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 JOsteopor,2016,22(1):49-52.
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[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 CaMKⅡδmediated 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 CaMKⅡδat dif ferent stages of osteoclast differentiation[J].Chin J Pathophysiol,2016,32(10):1870-1874.
[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 m RNA 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.
[6]Park KH,Gu DR,Jin SH,et al.Pueraria lobate inhibits RANKL-mediated osteoclastogenesis via downregulation 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ⅡδRNA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 CaMKⅡδRNA 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 downregulating 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 JOsteopor,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 CaMKⅡδmediated 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 CaMKⅡδat dif ferent stages of osteoclast differentiation[J].Chin J Pathophysiol,2016,32(10):1870-1874.
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