Zhong Xiaohuan, Wang Huixin
Center of Stomatology, Xiangya Hospital, Central South University, Changsha, China.
J Periodontal Res. 2022 Dec;57(6):1148-1158. doi: 10.1111/jre.13052. Epub 2022 Sep 5.
Periodontal ligament cells (PDLCs) possess the capacity to differentiate into a variety of cell types to benefit periodontal regeneration. In this study, we examined the circSKIL/miR-532-5p/Notch1 axis in controlling the osteoblastic differentiation of PDLCs.
Primary human PDLCs (hPDLCs) were isolated and induced to differentiate into osteoblasts. Osteogenic responses were assessed for the expressions of osteoblast-related marker proteins (including alkaline phosphatase (ALP), osteocalcin (OCN), bone morphogenetic protein-2 (BMP2), and runt-related transcription factor 2 (RUNX2) by RT-PCR. The formation of mineralized nodules was examined by Alizarin Red S (ARS) staining and ALP activity. Expressions of circSKIL, miR-532-5p, and Notch1 were measured by RT-PCR and western blotting, and their regulations by combining bioinformatic analysis and luciferase reporter assay. Notch signaling was assessed for the expressions of hairy and enhancer of split-1 (HES1) and Notch intracellular domain (NICD).
During osteoblastic differentiation of hPDLCs, circSKIL, and Notch1 were up-regulated, while miR-532-5p down-regulated. By sponging miR-532-5p, circSKIL activated Notch signaling, increasing levels of Notch1, HES1, and NICD. Functionally, knocking down circSKIL or overexpressing miR-532-5p inhibited osteoblastic differentiation of PDLCs, down-regulating ALP, OCN, BMP2, and RUNX2, and reducing ARS staining or ALP activity. The impacts of circSKIL knockdown were rescued by miR-532-5p inhibitor or overexpressing Notch1, while those caused by up-regulating miR-532-5p were reversed by overexpressing Notch1.
By targeting miR-532-5p and up-regulating Notch1, circSKIL critically controls osteoblastic differentiation of hPDLCs. Therefore, modulating this axis may maximize the differentiation of PDLCs into osteoblasts and benefit periodontal regeneration.
牙周膜细胞（PDLCs）具有分化为多种细胞类型以促进牙周组织再生的能力。在本研究中，我们检测了环状SKIL/miR-532-5p/Notch1轴在调控PDLCs成骨分化中的作用。
分离原代人PDLCs（hPDLCs）并诱导其分化为成骨细胞。通过RT-PCR检测成骨相关标志物蛋白（包括碱性磷酸酶（ALP）、骨钙素（OCN）、骨形态发生蛋白-2（BMP2）和 runt相关转录因子2（RUNX2））的表达，以评估成骨反应。通过茜素红S（ARS）染色和ALP活性检测矿化结节的形成。通过RT-PCR和蛋白质印迹法检测环状SKIL、miR-532-5p和Notch1的表达，并结合生物信息学分析和荧光素酶报告基因检测来研究它们之间的调控关系。通过检测毛状分裂增强子1（HES1）和Notch胞内结构域（NICD）的表达来评估Notch信号通路。
在hPDLCs成骨分化过程中，环状SKIL和Notch1表达上调，而miR-532-5p表达下调。环状SKIL通过吸附miR-532-5p激活Notch信号通路，增加Notch1、HES1和NICD的水平。在功能上，敲低环状SKIL或过表达miR-532-5p可抑制PDLCs的成骨分化，下调ALP、OCN、BMP2和RUNX2的表达，并降低ARS染色或ALP活性。miR-532-5p抑制剂或过表达Notch1可挽救环状SKIL敲低的影响，而过表达Notch1可逆转上调miR-532-5p所产生的影响。
环状SKIL通过靶向miR-532-5p并上调Notch
