Applied Oral Sciences & Community Dental Care, Faculty of Dentistry, The University of Hong Kong, Sai Ying Pun, Hong Kong SAR; Department of Orthodontics, School and Hospital of Stomatology, Cheeloo College of Medicine, Shandong University & Shandong Key Laboratory of Oral Tissue Regeneration & Shandong Engineering Research Center of Dental Materials and Oral Tissue Regeneration & Shandong Provincial Clinical Research Center for Oral Diseases, Jinan, Shandong, China.
Applied Oral Sciences & Community Dental Care, Faculty of Dentistry, The University of Hong Kong, Sai Ying Pun, Hong Kong SAR.
Int Dent J. 2024 Oct;74(5):1110-1119. doi: 10.1016/j.identj.2024.03.004. Epub 2024 Mar 28.
Specific circular RNAs (circRNAs) have been proven to play crucial roles in osteogenesis in vitro and in vivo. This study aims to identify a certain circRNA involved in the osteogenic differentiation of periodontal ligament stem cells (PDLSCs) and explore its regulatory role.
The expression of 5 candidate circRNAs (circ_0026344, circ_ACAP2, circ_0003764, circ_0008259, and circ_0060731) was detected by real-time quantitative reverse-transcription polymerase chain reaction (qRT-PCR) after PDLSCs were cultured in the osteogenic induction medium or medium supplemented with tumour necrosis factor-α (TNF-α, 10 ng/mL) for 3 and 7 days. The circRNA significantly decreased in both 3 and 7 days of osteogenic induction in PDLSCs and markedly increased in TNF-α-induced PDLSCs for 3 and 7 days screened. Identified circRNA was knocked down or overexpressed, and the effect on the osteogenic differentiation of PDLSCs was investigated by qRT-PCR, western blot, alkaline phosphatase (ALP) staining, and alizarin red S (ARS) staining. Cell counting kit-8 (CCK-8) assay and 5-ethynyl-2'-deoxyuridine (EdU) assay were applied to detect the effect of the circRNA on the proliferation of PDLSCs.
qRT-PCR results showed that the expression of circ_0003764 was significantly decreased when PDLSCs were cultured in the osteogenic induction medium for 3 or 7 days, whereas it was dramatically increased in TNF-α-induced PDLSCs. Knockdown of circ_0003764 promoted the expression of the osteogenesis-related genes (RUNX2, ALP, OCN) and proteins (RUNX2, OCN), enhanced the ALP activity, and elevated the mineralization by PDLSCs, as shown by ARS staining. However, with the overexpression of circ_0003764, the osteogenic differentiation capacity of PDLSCs was significantly reduced. The CCK-8 and EdU results indicated that circ_0003764 could inhibit the proliferation of PDLSCs.
Circ_0003764 is involved in the osteogenesis process and inhibits the osteogenic differentiation and proliferation of PDLSCs.
This study indicates that circ_0003764 can serve as a diagnostic and therapeutic target in bone regeneration-related diseases treated by PDLSCs-based tissue engineering.
特定的环状 RNA（circRNA）已被证明在体外和体内的成骨中发挥关键作用。本研究旨在鉴定一种参与牙周膜干细胞（PDLSCs）成骨分化的特定 circRNA，并探讨其调节作用。
通过实时定量逆转录聚合酶链反应（qRT-PCR）检测 5 种候选 circRNA（circ_0026344、circ_ACAP2、circ_0003764、circ_0008259 和 circ_0060731）在 PDLSCs 培养于成骨诱导培养基或补充肿瘤坏死因子-α（TNF-α，10ng/mL）的培养基中 3 天和 7 天后的表达。筛选出在 PDLSCs 成骨诱导的 3 天和 7 天内均显著降低，而在 TNF-α诱导的 PDLSCs 中 3 天和 7 天内显著增加的 circRNA。敲低或过表达鉴定出的 circRNA，通过 qRT-PCR、western blot、碱性磷酸酶（ALP）染色和茜素红 S（ARS）染色检测其对 PDLSCs 成骨分化的影响。细胞计数试剂盒-8（CCK-8）检测和 5-乙炔基-2'-脱氧尿苷（EdU）检测应用于检测 circRNA 对 PDLSCs 增殖的影响。
qRT-PCR 结果显示，当 PDLSCs 培养于成骨诱导培养基中 3 天或 7 天时，circ_0003764 的表达明显降低，而在 TNF-α诱导的 PDLSCs 中则显著增加。circ_0003764 的敲低促进了成骨相关基因（RUNX2、ALP、OCN）和蛋白（RUNX2、OCN）的表达，增强了 PDLSCs 的 ALP 活性，并通过 ARS 染色提高了其矿化能力。然而，circ_0003764 的过表达显著降低了 PDLSCs 的成骨分化能力。CCK-8 和 EdU 结果表明，circ_0003764 可抑制 PDLSCs 的增殖。
circ_0003764 参与成骨过程，并抑制 PDLSCs 的成骨分化和增殖。
本研究表明，circ_0003764 可作为基于牙周膜干细胞的组织工程治疗相关骨再生疾病的诊断和治疗靶点。
