Department of Oral and Maxillofacial Surgery, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, PR China; Guangdong Key Laboratory of Stomatology, Sun Yat-sen University, Guangzhou, PR China.
Department of Oral and Maxillofacial Surgery, Hainan General Hospital, Haikou, PR China.
Biochem Biophys Res Commun. 2018 Sep 5;503(2):815-821. doi: 10.1016/j.bbrc.2018.06.081. Epub 2018 Jun 22.
Long noncoding RNAs (lncRNAs) were proposed to be important regulators influencing various differentiation processes. Yet, the molecular mechanisms of lncRNAs governing osteogenic differentiation of Periodontal Ligament Stem Cells (PDLSCs) remain unclear. Here, PDLSCs were isolated from normal periodontal ligament of human (PDL) whereas P-PDLSCs were isolated from periodontitis affected PDL. Quantitative real-time PCR (qRT-PCR) was performed to examine the relative expression level of lncRNA-ANCR and of Osterix (OSX), Alkaline Phosphatase (ALP) as well as Runt-related transcription factor 2 (RUNX2) in PDLSCs. Gain- and loss-of- function experiments was performed to study the role of lncRNA-ANCR. Alizarin Red staining was used to evaluate the function of lncRNA-ANCR and miRNA-758 on osteogenic differentiation. In addition, via dual luciferase reporter assay and RNA immunoprecipitation the microRNA sponge potential of lncRNA-ANCR was assessed. A luciferase reporter assay identified the correlation between miR-758 and Notch2. Our results showed that the expression of ALP, RUNX2 and OSX were increased whereas lncRNA-ANCR was decreased during the process of differentiation in PDLSCs. Overexpression of lncRNA-ANCR decreased the expression of ALP, RUNX2 and OSX as confirmed by Alizarin red staining. Overexpression of lncRNA-ANCR resulted in reduction of the miR-758 expression level. Furthermore, RNA immunoprecipitation proved that lncRNA-ANCR targets miR-758 directly. The results of dual luciferase reporter assay also demonstrated that miR-758 regulated Notch2 expression by targeting 3'-UTR of Notch2. In conclusion, the novel pathway lncRNA-ANCR/miR-758/Notch2 plays an important role in the process of regulating osteogenic differentiation of PDLSCs.
长链非编码 RNA（lncRNA）被认为是影响各种分化过程的重要调节因子。然而，lncRNA 调控牙周膜干细胞（PDLSCs）成骨分化的分子机制尚不清楚。本研究从正常人牙周膜（PDL）中分离出牙周膜干细胞（PDLSCs），从牙周炎影响的牙周膜（PDL）中分离出 P-PDLSCs。采用实时定量 PCR（qRT-PCR）检测 lncRNA-ANCR 和 Osterix（OSX）、碱性磷酸酶（ALP）以及 Runt 相关转录因子 2（RUNX2）在 PDLSCs 中的相对表达水平。进行 gain- 和 loss-of- function 实验以研究 lncRNA-ANCR 的作用。采用茜素红染色评估 lncRNA-ANCR 和 miRNA-758 对成骨分化的作用。此外，通过双荧光素酶报告基因检测和 RNA 免疫沉淀评估 lncRNA-ANCR 的 miRNA 海绵潜力。荧光素酶报告基因检测鉴定了 miR-758 与 Notch2 之间的相关性。结果显示，在 PDLSCs 分化过程中，ALP、RUNX2 和 OSX 的表达增加，而 lncRNA-ANCR 的表达减少。lncRNA-ANCR 的过表达通过茜素红染色证实降低了 ALP、RUNX2 和 OSX 的表达。lncRNA-ANCR 的过表达导致 miR-758 的表达水平降低。此外，RNA 免疫沉淀证实 lncRNA-ANCR 直接靶向 miR-758。双荧光素酶报告基因检测结果还表明，miR-758 通过靶向 Notch2 3'-UTR 调节 Notch2 表达。综上所述，lncRNA-ANCR/miR-758/Notch2 的新途径在调节 PDLSCs 成骨分化过程中发挥重要作用。
