环状 RNA SIPA1L1 通过调控牙髓干细胞中 miR-617/Smad3 轴促进成骨。
Circular RNA SIPA1L1 promotes osteogenesis via regulating the miR-617/Smad3 axis in dental pulp stem cells.
机构信息
Institute of Stomatology, Nanjing Medical University, 136 Hanzhong Road, Nanjing, 210029, Jiangsu, China.
Key Laboratory of Oral Diseases of Jiangsu Province and Stomatological Institute of Nanjing Medical University, 140 Hanzhong Road, Nanjing, 210029, Jiangsu, China.
出版信息
Stem Cell Res Ther. 2020 Aug 24;11(1):364. doi: 10.1186/s13287-020-01877-3.
BACKGROUND
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.
METHODS
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.
RESULTS
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.
CONCLUSION
CircRNASIPA1L1 promotes osteogenesis of DPSCs by adsorbing miR-617 and further targeting Smad3.
背景
骨再生是治疗肿瘤、骨缺损、骨折等引起的骨丢失的首选方法。最近，间充质干细胞被认为是骨缺损治疗的有希望的工具。牙髓干细胞（DPSCs）是再生医学和骨再生的有前途的候选者。我们之前的研究表明，DPSCs 成骨过程中 circSIPA1L1 的上调具有重要意义。在本文中，探讨了 circSIPA1L1 在 DPSCs 成骨中的潜在作用及其潜在机制。
方法
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 DPSCs 矿化沉积的调节作用，并进行体内骨形成实验验证 circSIPA1L1 对 DPSCs 的生物学影响。通过 Western blot 检测 Smad3 蛋白表达。通过 FISH 证实 circSIPA1L1 和 miR-617 的定位。通过双荧光素酶报告基因实验和挽救实验研究 circSIPA1L1/miR-617/Smad3 调控轴在 DPSCs 成骨中的作用。
结果
Sanger 测序和背靠背引物实验证实了 circSIPA1L1 的闭环结构。CircSIPA1L1 可促进 DPSCs 的定向分化。通过 MiRDB、miRTarBase 和 TargetScan 数据库分析预测 miR-617 是 circSIPA1L1 的靶结合物，通过双荧光素酶报告基因实验进一步证实。FISH 结果显示 circSIPA1L1 和 miR-617 在 DPSCs 的细胞质中共定位。miR-617 对 DPSCs 的成骨作用具有抑制作用。circSIPA1L1 敲低或 miR-617 上调均下调磷酸化 Smad3。此外，挽救实验表明，circSIPA1L1 敲低逆转了 miR-617 对 DPSCs 成骨的抑制作用。
结论
CircRNASIPA1L1 通过吸附 miR-617 促进 DPSCs 成骨，并进一步靶向 Smad3。
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