hDPSCs, a type of MSCs obtained from dental pulp tissues, have the potential to differentiate into odontoblasts and osteoblasts in vitro and in vivo [1, 19, 20]. Compared to BMSCs, hDPSCs are more easily accessible, and exhibits stronger proliferative activity due to their higher expression of cyclin-dependent kinase 6 and cell cycle mediators such as insulin-like growth factor [21]. For these reasons, hDPSCs have been considered as a promising candidate for dental tissue regeneration and bone defect repair. Regardless of the tissue source, MSCs possess the capacity of self-renewal and multidirectional differentiation, and share similar cellular surface markers [19, 20]. In this study, the isolated hDPSCs positively expressed MSC-specific cell surface markers CD44, CD73 and CD90 and negatively expressing hematopoietic surface markers CD45, and were capable of differentiating into osteoblasts and adipocytes.
Among of lncRNAs, the TUG1 has shown to be involved in the regulation of several cellular behaviors and its dysregulation could resulted in a myriad of diseases, such as cancer, cardiovascular disease and diabetic complications [7, 22–24]. The expression level of TUG1 was significantly increased in various types of cancer [7, 22]. In addition, recent studies have demonstrated the important role of TUG1 in osteogenic differentiation. For insurance, Teng et al. found that the TUG1 was downregulated in osteoporosis and affected the osteogenesis of BMSCs [9]. TUG1 also promoted osteoblast differentiation by sponging miR-204-5p [25]. In another study, He et al. reported that TUG1 facilitated osteogenesis of periodontal ligament stem cells by targeting Lin28A [10]. All of these indicate that TUG1 plays a positive role during osteogenic differentiation. However, whether it also promotes osteo/odontogenesis of hDPSCs has not been reported yet.
In this study, the expression of TUG1 was upregulated during osteo/odontogenic induction of hDPSCs. Silencing TUG1 inhibited both the early ALP activity and late mineralized nodule formation in the process of osteo/odontogenesis of hDPSCs. DSPP is a pre-protein secreted by odontoblasts. Its proteolytic cleavage products, dentin sialoprotein and dentin phosphoprotein, are widely present in the extracellular matrix of dental pulp cells [26, 27]. DMP1 mainly express in the initial stages of odontogensis, which has been considered as an early marker of odontoblast differentiation [27, 28]. Both of the DSPP and DMP1 play important roles in the mineralization of dentin and bone [26]. RUNX2 is a master organizer of gene transcription during the differentiation of osteoblasts. RUNX2 mainly expresses in the initial stage of osteogenesis and has been shown to directly activate the expression of many genes required for osteoblast differentiation and bone matrix production, including OCN, OPN, bone sialoprotein, and collagen I [29, 30]. In our study, we found that the expression of DSPP, DMP1, RUNX2, OCN and OPN were significantly decreased in TUG1-silenced hDPSCs. The results indicated that the TUG1 also plays an accelerative role during osteo/odontogenic differentiation in hDPSCs, consistent with most previous reports. However, the role of TUG1 in osteogenic differentiation remains an area of debate. In Zhang et al.’s study, the TUG1 was reported to suppress the osteogenic differentiation of irradiation treated BMSCs via Smad5 [31]. This discrepancy may be attributed to the diverse functions of TUG1 in interacting with distinct targets under specific conditions.
The importance of canonical Wnt/β-catenin signaling in bone homeostasis has been highlighted by a large number of studies. Mutations within this signaling pathway can result in abnormalities of bone mass, with inhibition leading to osteoporosis and activation leading to high bone mass phenotypes [14, 32]. Activation of Wnt/β-catenin signaling in osteocytes promoted the osteogenic differentiation of BMSCs [33]. Wnt/β-catenin signaling also plays a crucial role in osteo/odontogenic differentiation of hDPSCs [34–36]. The Wnt/β-catenin signaling was activated in the hypoxia-inducible factor 1α (HIF1α) induced osteo/odontogenic differentiation of hDPSCs [35]. Gong et al. discovered that the R-spondin 2 promote osteo/odontogenic differentiation of hDPSCs in cooperation with Wnt3a [36]. In our study, silencing of TUG1 resulted in inhibition of the Wnt/β-catenin signaling pathway during the osteo/odontogenic development of hDPSCs. Furthermore, rescue experiments showed that LiCl could reverse the inhibiting effect of TUG1 silencing on osteo/odontogenic differentiation.
Recently, increasing evidence showed that lncRNAs played important roles in osteo/odontogenic differentiation of hDPSCs. Several lncRNAs have been found to regulate this process through different mechanisms. For instance, lncRNA SNHG7 could promote the osteo/odontogenic differentiation of hDPSCs by sponging miR-6512-3p [37]. Through enhancing the protein level and demethylase activity of JMJD3, lncRNA MALAT1 decreased the H3K27me3 occupancy of the promoter region of DSPP and DMP1, which ultimately promoted their expression [38]. Interestingly, two independent studies by Du et al. and Zhong et al. have revealed distinct mechanisms by which lncRNA H19 facilitates odontogenic differentiation of hDPSCs [39, 40]. These results suggest that the regulatory mechanisms of lncRNAs in osteo/odontogenic differentiation of hDPSCs are complex and diverse. To the best of our knowledge, our study is the first report about the role of TUG1 in osteo/odontogenesis of hDPSCs. We found that TUG1 promoted osteo/odontogenic differentiation, which at least partially by Wnt/β-catenin signaling. However, there were some shortcomings to our study. Firstly, it is debatable about the role of the Wnt/β-catenin signaling pathway in hDPSCs osteo/odontogenic differentiation [35, 41, 42]. According to Fu et al.’s study, lncRNA SNHG1 promoted odontogenic induction of hDPSCs through inhibiting Wnt/β-catenin pathway [42]. Secondly, the exact mechanism by TUG1 regulates Wnt/β-catenin signaling remains obscure. Furthermore, whether there are other mechanisms of TUG1 in regulating hDPSCs osteo/odontogenic differentiation. All of these are warranted to address in future studies, which can advance our understanding on lncRNAs regulating osteo/odontogenic differentiation in hDPSCs.