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

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。

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

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.