CaMKⅡγ基因沉默对破骨细胞分化中NFATc1、TRAP、c-Src基因表达的影响

doi:10.11904/j.issn.1002-3070.2019.04.002

实用肿瘤学杂志 ›› 2019, Vol. 33 ›› Issue (4): 294-299.doi: 10.11904/j.issn.1002-3070.2019.04.002

CaMKⅡγ基因沉默对破骨细胞分化中NFATc1、TRAP、c-Src基因表达的影响

刘梦楠¹, 王会¹, 戚孟春¹, 董伟¹, 李任¹, 孙红²

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

收稿日期:2019-02-18 发布日期:2019-08-30
通讯作者: 戚孟春,E-mail:qimengchun@163.com
作者简介:刘梦楠,女,(1991-),硕士研究生,从事口腔颌面外科学的研究。
基金资助:
国家自然科学基金(编号:81270965);河北省教育厅重点研究项目(编号:ZD2015005);河北省自然科学基金(编号:H2017209114)

Effect of CaMKIIγ RNA interference on the expression of NFATc1,TRAP and c-Src genes in osteoclast differentiation

LIU Mengnan¹, WANG Hui¹, QI Mengchun¹, DONG Wei¹, LI Ren¹, SUN Hong²

1.Department of Oral and Maxillofacial Surgery,College of Stomatology,North China University of Science and Technology,Tangshan 063000,China;
2.Department of Pathology,College of Basic Medicine,North China University of Science and Technology

Received:2019-02-18 Published:2019-08-30

摘要/Abstract

摘要： 目的研究钙调蛋白依赖性激酶II(Calmodulin-dependent kinase II,CaMKII)γ基因沉默对破骨细胞分化过程中活化T-细胞核因子c1(Nuclear factor of activated T-cells cytoplasmic 1,NFATc1)、非受体酪氨酸激酶(Cell-sarcoma receptor coactivator,c-Src)、抗酒石酸酸性磷酸酶(Tartrate resistant acid phosphatase,TRAP)基因表达的影响,探讨CaMKIIγ在破骨细胞分化中的作用和分子机制。方法用慢病毒构建CaMKIIγ RNA干扰载体,转染RAW264.7细胞,实验分为A、B、C三组,分别为对照组、阴性载体组和干扰载体组;三组细胞转染12 h后,用50 ng RANKL诱导向破骨细胞分化;诱导5 d后收获细胞,采用实时荧光定量 PCR、Western blot、免疫荧光化学检测三组细胞中NFATc1、TRAP、c-Src基因表达情况。结果 C组中NFATc1、TRAP、c-Src mRNA水平较A组分别下降了49.86%、43.65%和53.57%(P＜0.001),蛋白水平分别下降了54.22%、46.75%和45.86%(P＜0.001);而B组与A组比较无统计学差异(P＞0.05)。免疫荧光化学检测C组上述基因荧光强度明显弱于A、B组,且破骨细胞生成明显少于A、B组。结论 CaMKIIγ RNA干扰显著抑制了NFATc1、TRAP、c-Src基因表达,提示CaMKIIγ在破骨细胞分化中起着关键调控作用。

关键词: 钙调蛋白依赖性激酶IIγ, RNA干扰, 活化T细胞核因子c1, 非受体酪氨酸激酶, 抗酒石酸酸性磷酸酶

Abstract: Objective The aim of this study was to investigate the effect of Ca²⁺/calmodulin-dependent kinase II(CaMKII)γ RNA interference on the expression of nuclear factor of activated T-cells cytoplasmic 1(NFATc1),tyrosine kinase(c-Src)and tartrate resistant acid phosphatase(TRAP)genes,and its role and molecular mechanism in osteoclast differentiation.Methods The CaMKII γ RNA interference vector was constructed by lentivirus and transfected into RAW264.7 cells.The experiment was divided into three groups:A,B and C,which were the control group,negative vector group and interference vector group.After transfection for 12 hours,osteoclasts induced by 50 ng/mL RANKL and the cells were harvested after induction for 5 days.Real-time quantitative PCR,Western blot and immunofluorescence were used to detect the expression of NFATc1,TRAP and c-Src genes in three groups.Results The mRNA levels of NFATc1,TRAP and c-Src in the group C decreased by 49.86%,43.65% and 53.57%,respectively(P＜0.001),and the protein levels decreased by 54.22%,46.75% and 45.86%,respectively(P＜0.001).There was no significant difference between the A and the B groups(P＞0.05).The fluorescence intensity of the above genes in the group C was significantly weaker than that in the A and B groups,and the formation of osteoclasts was significantly less than that in the A and B groups.Conclusion CaMKIIγ RNA interference significantly inhibited the expression of NFATc1,TRAP and c-Src genes,suggesting that CaMKIIγ plays a key regulatory role in osteoclast differentiation.

Key words: Calmodulin-dependent kinase IIγ, RNA interference, Nuclear factor of activated T-cells cytoplasmic 1, Cell-sarcoma receptor coactivator, Tartrate resistant acid phosphatase

中图分类号:

R782

刘梦楠, 王会, 戚孟春, 董伟, 李任, 孙红. CaMKⅡγ基因沉默对破骨细胞分化中NFATc1、TRAP、c-Src基因表达的影响[J]. 实用肿瘤学杂志, 2019, 33(4): 294-299.

LIU Mengnan, WANG Hui, QI Mengchun, DONG Wei, LI Ren, SUN Hong. Effect of CaMKIIγ RNA interference on the expression of NFATc1,TRAP and c-Src genes in osteoclast differentiation[J]. Journal of Practical Oncology, 2019, 33(4): 294-299.

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CaMKⅡγ基因沉默对破骨细胞分化中NFATc1、TRAP、c-Src基因表达的影响

Effect of CaMKIIγ RNA interference on the expression of NFATc1,TRAP and c-Src genes in osteoclast differentiation

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