Long noncoding RNA TP53TG1 promotes odontogenic and osteogenic differentiation of stem cells from the apical papilla

doi:10.12307/2025.563

Abstract

Abstract: BACKGROUND: Long noncoding RNA TP53TG1 (lncRNA TP53TG1) is involved in regulating the proliferation, migration, invasion, and apoptosis of various cancer cells, but there are few reports on its role in other cells.
OBJECTIVE: To investigate the effects and pathways of lncRNA TP53TG1 on the proliferation and differentiation of human stem cells from the apical papilla.
METHODS: Human stem cells from the apical papilla were isolated and cultured, and then transfected with lncRNA TP53TG1 overexpression lentivirus. RT-qPCR was used to detect the overexpression efficiency of lncRNA TP53TG1. Western blot assay was used to detect the relative expression levels of PI3K, AKT, ERK, P38, Smad3, and their phosphorylated proteins. Human stem cells from the apical papilla were divided into the empty lentiviral vector transfection group and the lncRNA TP53TG1 overexpression group. CCK-8 assay was used to measure the cell proliferation. Alkaline phosphatase activity was detected by alkaline phosphatase staining on day 5 of osteogenic induction. Formation of mineralized nodules was detected by alizarin red staining on day 21 of osteogenic induction. RT-qPCR was used to detect the mRNA expression levels of dentin sialophosphoprotein, Runt-related transcription factor 2, dentin matrix protein 1, and bone sialoprotein on days 3, 7, and 14 of osteogenic induction. Western blot assay was used to detect the protein expression levels of dentin sialophosphoprotein and Runt-related transcription factor 2 on days 3, 7, and 14 of osteogenic induction.
RESULTS AND CONCLUSION: (1) RT-qPCR results showed that the lentivirus was successfully integrated into the genome of stem cells from the apical papilla. Western blot assay results showed that overexpression of lncRNA TP53TG1 up-regulated the protein levels of p-PI3K and p-AKT without affecting the expression of phosphorylated proteins in other pathways. (2) Starting from day 3 of cell culture, overexpression of lncRNA TP53TG1 significantly promoted the proliferation of stem cells from the apical papilla. (3) In the process of inducing odontogenic differentiation of stem cells from the apical papilla, overexpression of lncRNA TP53TG1 promoted the expression of odontogenic and osteogenic differentiation-related genes and proteins, significantly increased alkaline phosphatase activity and mineralized nodule formation. (4) The results show that lncRNA TP53TG1 may promote the odontogenic and osteogenic differentiation of stem cells from the apical papilla by activating the PI3K/AKT signaling pathway.

Key words: ">stem cell from the apical papilla, long noncoding RNA, lncRNA TP53TG1, virus transfection, dentinogenesis, PI3K/AKT signaling pathway, engineered stem cell

CLC Number:

Li Tingyue, Guo Qian, He Wenxi, Wu Jiayuan. Long noncoding RNA TP53TG1 promotes odontogenic and osteogenic differentiation of stem cells from the apical papilla[J]. Chinese Journal of Tissue Engineering Research, 2025, 29(36): 7776-7782.

Figures/Tables 2

2.1 根尖牙乳头干细胞的分离培养及鉴定结果收集根尖孔未闭合完全的年轻恒牙，根尖部可见质地坚韧的根尖牙乳头组织(图1A)。组织块消化培养5 d后，镜下可见梭形细胞从组织块中央呈放射状爬出，即为原代根尖牙乳头干细胞(图1B)。根尖牙乳头干细胞经成骨诱导21 d后，茜素红染色镜下可观察到红褐色矿化结节沉积(图1C)；经成脂诱导21 d后，油红O染色镜下可见红色脂滴形成(图1D)，表明获得的根尖牙乳头干细胞具有多向分化潜能。流式细胞术结果显示，根尖牙乳头干细胞高表达间充质干细胞标志物CD29、CD105、CD90和STRO-1，而不表达造血干细胞标志物CD34、CD45(图1E)，符合根尖牙乳头干细胞免疫表型特点。 2.2 慢病毒转染诱导根尖牙乳头干细胞中lncRNA TP53TG1的表达利用慢病毒转染技术，将携带绿色荧光标签的空载体慢病毒或过表达lncRNA TP53TG1的慢病毒转染至根尖牙乳头干细胞，24 h后通过嘌呤霉素筛选慢病毒感染成功的根尖牙乳头干细胞，72 h后通过荧光显微镜观察到两组均强烈表达绿色荧光(图2A，B)。此外，相比于空载体慢病毒组，lncRNA TP53TG1过表达组根尖牙乳头干细胞中lncRNA TP53TG1 mRNA表达显著增高(图2C，P < 0.001)，可用于后续实验。 2.3 过表达lncRNA TP53TG1促进根尖牙乳头干细胞增殖和成骨分化使用CCK-8法检测过表达lncRNA TP53TG1对根尖牙乳头干细胞增殖的影响。结果显示，从第3天开始，过表达lncRNA TP53TG1显著增强根尖牙乳头干细胞的增殖能力(图3A，P < 0.000 1)。碱性磷酸酶染色结果显示，与空载体组相比，过表达lncRNA TP53TG1组形成颜色更深的蓝紫色沉淀(图3B)，碱性磷酸酶活性检测结果也显示过表达lncRNA TP53TG1显著增强根尖牙乳头干细胞中碱性磷酸酶活性(图3C，P < 0.001)。茜素红染色结果显示lncRNA TP53TG1过表达组根尖牙乳头干细胞形成更多的红褐色矿化结节(图3D，E，P < 0.000 1)。 "

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