口腔医学研究

口腔医学研究 ›› 2017, Vol. 33 ›› Issue (6): 658-662.DOI: 10.13701/j.cnki.kqyxyj.2017.06.020

• 临床研究论著 • 上一篇    下一篇

miR-30a在雷帕霉素刺激人炎症牙周膜干细胞后的表达情况及其与Beclin1基因的靶向调控作用的研究

邵江红1, 陈芳1, 孔宇2*   

  1. 1. 首都医科大学附属北京康复医院口腔科 北京 100144;
    2. 北京大学口腔医院综合科 北京 100081)
  • 收稿日期:2016-12-15 出版日期:2017-06-20 发布日期:2017-06-26
  • 通讯作者: 孔宇,电话:010-62179977-5592
  • 作者简介:邵江红(1973~ ),女,江西余干人,学士,主治医师,主要从事口腔修复及牙周的临床治疗工作。

Effects of Rapamycin on Expression of MiR-30a in Inflammatory PDLSCs and Research on MicroRNA-30a Targeted Regulation of Beclin1 Gene Expression.

SHAO Jiang-hong1, CHEN Fang1, KONG Yu2*.   

  1. 1. Beijing Rehabilitation Hospital of Capital Medical University, Department of Stomatology, Beijing 100144, China;
    2. Peking University Stomatological Hospital, Department of General Dentistry, Beijing 100081, China.
  • Received:2016-12-15 Online:2017-06-20 Published:2017-06-26

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摘要： 目的:研究雷帕霉素对人牙周膜干细胞中miR-30a的表达的影响,并验证其与Beclin1的靶向调控关系,探讨miR-30a与自噬途径在牙周膜干细胞的炎症作用机制。方法:分离培养人牙周膜干细胞,并分别进行50 μg/L雷帕霉素作用2 h,10 nmol/L 3-甲基腺嘌呤(3-MA)作用12 h,收集细胞,提取总RNA;应用实时荧光定量PCR(qRT-PCR)检测miR-30a的表达水平;应用qRT-PCR、Western blot及双荧光素酶报告系统验证miR-30a与Beclin1的相互作用关系。结果:雷帕霉素刺激细胞后,细胞内的miR-30a的表达升高;qRT-PCR、Western blot及双荧光素酶报告系统证实,miR-30a对Beclin1的mRNA及蛋白均有抑制,且与Beclin1的3’UTR具有靶向抑制作用。结论:miR-30a可靶向抑制自噬相关基因Beclin1的表达,并参与人牙周膜干细胞的细胞自噬过程。

关键词: MiRNA-30a, 牙周膜干细胞, Beclin1, 细胞自噬

Abstract: Objective: To study the effect of rapamycin on expression of miR-30a in inflammatory periodontal ligament cells (IPDLSCs), verify the relationship of miR-30a and Beclin1, and study the mechanism of miR-30a on IPDLSCs autophagy. Methods: Cells were isolated from inflammatory periodontal ligament samples and cultured with 50ng/ml rapamycin (2 hours) and 10 nmol/L 3-methyl adenine (3-MA,12 hours), respectively. The cells were collected for total RNA. The expression levels miR-30a was detected by real-time PCR. The effect of miR-30a on Beclin1 was verified by real-time PCR, Western blot and the dual-luciferase reporter assay. Results: The expression of miR-30a in IPDLSCs treated with rapamycin was higher than that of group without rapamycin. The real-time PCR, Western blot, and the dual-luciferase reporter assay system demonstrated that miR-30a could suppress Beclin1 expression of mRNA and protein by targeting the specific 3’ untranslated region (3’ UTR) sequence of Beclin1 gene. Conclusion: MiR-30a can directly inhibit the autophagy-related gene Beclin1 expression and participate in the regulation of autophagy process in IPDLSCs.

Key words: MiRNA-30a , PDLSCs , Beclin1 , Cell autophagy

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