Bone regeneration is preferred for bone loss caused by tumors, bone defects, fractures, etc. Recently, mesenchymal stem cells are considered as optimistic tools for bone defect therapy. Dental pulp stem cells (DPSCs) are a promising candidate for regenerative medicine and bone regeneration. Our previous study showed that upregulated circSIPA1L1 during osteogenesis of DPSCs is of significance. In this paper, the potential role of circSIPA1L1 in osteogenesis of DPSCs and its underlying mechanisms are explored. The circular structure of circSIPA1L1 was identified by Sanger sequencing and PCR. Regulatory effects of circSIPA1L1 and miR-617 on mineral deposition in DPSCs were assessed by alkaline phosphatase (ALP) and alizarin red S (ARS) staining and in vivo bone formation assay were conducted to verify the biological influences of circSIPA1L1 on DPSCs. Western blot was performed to detect the protein expression of Smad3. Localization of circSIPA1L1 and miR-617 was confirmed by FISH. Dual-luciferase reporter assay and rescue experiments were conducted to investigate the role of the circSIPA1L1/miR-617/Smad3 regulatory axis in osteogenesis of DPSCs. Sanger sequencing and back-to-back primer experiments confirmed the closed-loop structure of circSIPA1L1. CircSIPA1L1 could promote the committed differentiation of DPSCs. MiR-617 was predicted to be the target binding circSIPA1L1 through MiRDB, miRTarBase, and TargetScan database analyses, which was further confirmed by dual-luciferase reporter assay. FISH results showed that circSIPA1L1 and miR-617 colocalize in the cytoplasm of DPSCs. MiR-617 exerted an inhibitory effect on the osteogenesis of DPSCs. Knockdown of circSIPA1L1 or upregulation of miR-617 downregulated phosphorylated Smad3. In addition, rescue experiments showed that knockdown of miR-617 reversed the inhibitory effect of circSIPA1L1 on osteogenesis of DPSCs. CircRNASIPA1L1 promotes osteogenesis of DPSCs by adsorbing miR-617 and further targeting Smad3.

中文翻译：

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环状RNA SIPA1L1通过调节牙髓干细胞中的miR-617 / Smad3轴促进成骨。

骨再生对于因肿瘤，骨缺损，骨折等引起的骨丢失是优选的。近来，间充质干细胞被认为是骨缺损治疗的乐观工具。牙髓干细胞（DPSC）是再生医学和骨再生的有前途的候选者。我们先前的研究表明，在DPSC的成骨过程中circSIPA1L1上调具有重要意义。本文探讨了circSIPA1L1在DPSCs成骨中的潜在作用及其潜在机制。通过Sanger测序和PCR鉴定circSIPA1L1的环状结构。通过碱性磷酸酶（ALP）和茜素红S（ARS）染色评估circSIPA1L1和miR-617对DPSC中矿物质沉积的调节作用，并进行体内骨形成试验以验证circSIPA1L1对DPSC的生物学影响。进行蛋白质印迹以检测Smad3的蛋白表达。FISH证实了circSIPA1L1和miR-617的定位。进行了双重荧光素酶报告基因测定和拯救实验，以研究circSIPA1L1 / miR-617 / Smad3调控轴在DPSC的成骨中的作用。Sanger测序和背对背引物实验证实了circSIPA1L1的闭环结构。CircSIPA1L1可以促进DPSC的持续分化。通过MiRDB，miRTarBase和TargetScan数据库分析，预测MiR-617是靶结合circSIPA1L1，这已通过双荧光素酶报告基因分析进一步证实。FISH结果表明，circSIPA1L1和miR-617在DPSC的细胞质中共定位。MiR-617对DPSC的成骨具有抑制作用。敲低circSIPA1L1或上调miR-617下调磷酸化的Smad3。此外，救援实验表明，敲低miR-617可以逆转circSIPA1L1对DPSC骨形成的抑制作用。CircRNASIPA1L1通过吸附miR-617并进一步靶向Smad3来促进DPSC的成骨作用。