miR-34a对人牙周膜干细胞成骨分化的促进作用及其机制

doi:10.13481/j.1671-587X.20210604

摘要/Abstract

摘要： 目的

探讨微小RNA-34a（miR-34a）对人牙周膜干细胞（PDLSCs）成骨分化的影响，并阐明其作用机制。

方法

原代分离PDLSCs，诱导PDLSCs成骨分化，收集成骨分化后第0、7和14 天的PDLSCs。实时荧光定量PCR（RT-qPCR）法检测PDLSCs成骨分化细胞中碱性磷酸酶（ALP）、Runt相关转录因子2（Runx2）和骨钙素（OCN）mRNA表达水平，Western blotting法检测PDLSCs中解整合素金属蛋白酶10（ADAM10）蛋白表达水平。将PDLSCs分为空白对照组、空载组和miR-34a过表达组。RT-qPCR法检测各组PDLSCs中miR-34a表达水平，双荧光素酶报告系统验证miR-34a与ADAM10基因的靶向关系，Western blotting法检测各组PDLSCs中ADAM10蛋白表达水平。成骨诱导分化后，采用茜素红染色观察各组PDLSCs中矿化结节形成情况， RT-qPCR法检测各组PDLSCs中ALP、Runx2和OCN mRNA表达水平，比色法检测各组PDLSCs中ALP活性，Western blotting法检测各组PDLSCs中Notch1、Notch胞内结构域（NICD）和Hes1蛋白的表达水平。

结果

与成骨分化第0天比较，成骨分化第7和14天PDLSCs中miR-34a、ALP、Runx2和OCN mRNA表达水平逐渐升高（P<0.05），而ADAM10蛋白表达水平逐渐降低（P<0.05）。与对照组和空载组比较，miR-34a过表达组细胞中miR-34a表达水平明显升高（P<0.05），ADAM10蛋白表达水平明显降低（P<0.05）。双荧光素酶报告基因实验，PDLSCs中ADAM10是miR-34a的靶基因。与对照组和空载组比较， miR-34a过表达组PDLSCs中矿化结节形成数、ALP活性以及ALP、Runx2和OCN mRNA表达水平明显升高（P<0.05），Notch1、NICD和Hes1蛋白表达水平明显降低（P<0.05）。

结论

过表达miR-34a能通过靶向下调ADAM10进而抑制Notch信号通路活性，从而促进PDLSCs成骨分化。

关键词: 微小RNA-34a, 解整合素金属蛋白酶10, 信号通路, 牙周膜干细胞, 成骨分化

Abstract: Objective

To explore the effect of microRNA-34a（miR-34a）on the osteogenic differentiation of the human periodontal ligament stem cells （PDLSCs）， and to clarify its mechanism.

Methods

The primary cultured PDLSCs were isolated and induced osteogenic differentiation. Then the PDLSCs were collected 0， 7 and 14 d after osteogenic differentiation. The expression levels of alkaline phosphatase （ALP）， Runt-related transcription factor 2 （Runx2）， and osteocalcin （OCN） mRNA in the PDLSCs were detected by Real-time fluorescence quantitative PCR（RT-qPCR） method.The expression level of a disintegrin and metalloprotease 10（ADAM10） protein in the PDLSCs was detected by Western blotting method. The PDLSCs were divided into blank control group， vector group，and miR-34a over-expression group. The expression levels of miR-34a in the PDLSCs in various groups were detected by RT-qPCR method， the targeted relationship between miR-34a and ADAM10 genes was verified by dual luciferase report system， and the expression levels of ADAM10 protein in the PDLSCs in various groups were detected by Western blotting method. After the PDLSCs were differentiated by osteogenesis， the formations of mineralized nodules in the PDLSCs in various groups were observed by Alizarin red staining， the expression levels of ALP， Runx2， and OCN mRNA in the PDLSCs in various groups were detected by RT-qPCR method， the ALP activities in the PDLSCs in various groups were detected by colorimetric method， the expression levels of Notch1， Notch intracellular domain （NICD）， and Hes1 proteins in the PDLSCs in various groups were detected by Western blotting method.

Results

Compared with 0 d after osteogenic differentiation， the expression level of miR-34a and the expression levels of ALP， Runx2 and OCN mRNA in the PDLSCs at 7 and 14 d after osteogenic differentiation were gradually increased （P<0.05）， while the expression levels of ADAM10 protein were gradually decreased（P<0.05）.Compared with blank control group and vector group， the expression level of miR-34a in miR-34a over-expression group was significantly increased（P<0.05），while the expression level of ADAM10 protein was significantly decreased（P<0.05）. The results of dual luciferase reporter gene system showed that ADAM10 was a target gene of miR-34a in the PDLSCs. Compared with blank control group and vector group， the formation amount of calcified nodules， the ALP activity， and the expression levels of ALP， Runx2， and OCN mRNA in the PDLSCs in miR-34a over-expression group were significantly increased（P<0.05）， while the expression levels of Notch1， NICD， and Hes1 proteins were significantly decreased（P<0.05）.

Conclusion

Over-expression of miR-34a can inhibit the activity of Notch signaling pathway by targeting down-regulation of ADAM10， thus promote the osteogenic differentiation of the PDLSCs.

Key words: microRNA-34a, a disintegrin and metalloprotease 10, signal pathway, periodontal ligament stem cells, osteogenic differentiation

中图分类号:

R780.2

董霞,王训霞,杨芳. miR-34a对人牙周膜干细胞成骨分化的促进作用及其机制[J]. 吉林大学学报(医学版), 2021, 47(6): 1362-1370.

Xia DONG,Xunxia WANG,Fang YANG. Promotion effect of miR-34a on osteogenic differentiation of human periodontal ligament stem cells and its mechanism[J]. Journal of Jilin University(Medicine Edition), 2021, 47(6): 1362-1370.

图/表 10

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