Circular RNA SIPA1L1 promotes osteogenesis via regulating the miR-617/Smad3 axis in dental pulp stem cells
Circular RNA SIPA1L1 promotes osteogenesis via regulating the miR-617/Smad3 axis in dental pulp stem cells
复制标题
环状RNA SIPA1L1通过调节牙髓干细胞中的miR-617/Smad3轴促进成骨
DOI：
10.1186/s13287-020-01877-3
复制
发表时间：
2020-08-24
影响因子：
7.5
通讯作者：
Yu, Jinhua
中科院分区：
文献类型：
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作者：
Ge, Xingyun;Li, Zehan;Yu, Jinhua
背景骨再生是治疗肿瘤、骨缺损、骨折等骨丢失的首选方法。近年来，间充质干细胞被认为是骨缺损治疗的理想工具。牙髓干细胞(DPSCs)是一种很有前途的再生医学和骨再生候选细胞。我们先前的研究表明，SIPA1L1在DPSCs成骨过程中的上调具有重要意义。本文探讨了CircSIPA1L1在牙髓间充质干细胞成骨中的作用及其机制。方法用Sanger测序和聚合酶链式反应鉴定CircSIPA1L1的环状结构。用碱性磷酸酶(ALP)和茜素红S(ARS)染色检测CircSIPA1L1和miR-617对DPSCs矿物质沉积的调节作用，并通过体内成骨实验验证CircSIPA1L1对DPSCs的生物学影响。Western印迹法检测Smad3蛋白的表达。FISH证实了CircSIPA1L1和miR-617的定位。通过双荧光素酶报告实验和挽救实验，研究了SIPA1L1/miR-617/Smad3调节轴在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定向分化。通过MiRDB、miRTarBase和TargetScan数据库分析，预测miR-617是与SIPA1L1结合的靶标，双荧光素酶报告实验进一步证实了这一点。FISH结果显示，CircSIPA1L1和miR-617共定位于DPSCs的细胞质中。MIR-617对DPSCs的成骨有抑制作用。抑制SIPA1L1或上调miR-617下调磷酸化的Smad3。此外，抢救性实验表明，miR-617基因的敲除逆转了CircSIPA1L1对DPSCs成骨的抑制作用。结论CircRNASIPA1L1通过吸附miR-617进一步靶向Smad3促进DPSCs成骨。
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.