上海口腔医学

上海口腔医学 ›› 2025, Vol. 34 ›› Issue (5): 457-464.doi: 10.19439/j.sjos.2025.05.002

• 论著 • 上一篇    下一篇

hDPSCs中RNA结合蛋白Lin28对let-7家族调控的生物信息学方法分析

刘昕昕1,*, 李婉怡1,*, 孙梦馨1, 张雨欣1, 吴宇熙1, 段荣泉1,2#, 刘岩1,2#   

  1. 1.徐州医科大学,江苏 徐州 221004;
    2.徐州医科大学附属口腔医院,江苏 徐州 221002
  • 收稿日期:2024-06-20 修回日期:2024-08-17 出版日期:2025-10-25 发布日期:2025-10-31
  • 通讯作者: 段荣泉,E-mail：RongquanD@xzhmu.edu.cn;刘岩,E-mail：1563625177@qq.com。#共同通信作者
  • 作者简介:刘昕昕（2002-）,女,在读本科生,E-mail: 1328561771@qq.com;李婉怡（2003-）,女,在读本科生,E-mail: 2337123711@qq.com。*并列第一作者
  • 基金资助:
    江苏省卫生健康委科研项目（Z2023015）; 徐州市卫生健康委医学科技创新项目（XWKYHT20240121）

Bioinformatics-based approach to analyze the regulation of let-7 family by RNA-binding protein Lin28 in hDPSCs

Liu Xinxin1, Li Wanyi1, Sun Mengxin1, Zhang Yuxin1, Wu Yuxi1, Duan Rongquan1,2, Liu Yan1,2   

  1. 1. Xuzhou Medical University. Xuzhou 221004;
    2. Affiliated Stomatological Hospital of Xuzhou Medical University. Xuzhou 221002, Jiangsu Province, China
  • Received:2024-06-20 Revised:2024-08-17 Online:2025-10-25 Published:2025-10-31

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摘要： 目的:采用生物学信息方法分析RNA结合蛋白Lin28调控人牙髓干细胞（human dental pulp stem cell,hDPSCs）增殖的可能机制。方法:通过慢病毒介导稳定转染Lin28基因至hDPSCs,RNA结合蛋白免疫共沉淀（RNA binding protein immunoprecipitation,RIP）检测过表达Lin28富集的mRNA浓度和总量。RIP测序（RIP-seq）检测Lin28富集的mRNA和相关通路变化。RIP-PCR验证RIP-seq测序结果,Real-time PCR检测Lin28和let-7家族各成员miRNA表达水平,TargetScan（http://www.targetscan. org/vert_71/）预测let-7a相关靶基因mRNA及具体结合位点,Western 免疫印迹法检测let-7a相关靶基因IGF1R、IRS2和IGF2BPs蛋白表达水平的变化。结果:RNA结合蛋白Lin28作为一种重要的调节因子,在细胞生长发育、组织代谢等方面起着关键作用。过表达Lin28的hDPSCs中,pri-let-7a/b/c/d/e/f/i/98及pre-let-7a/b/c/d/e/f/i/98均有不同程度下降。Lin28过表达可上调let-7相关靶基因IGF2BP1和IGF2BP2蛋白表达水平。结论:Lin28在多条途径上通过特异性互补结合pri-let-7及pre-let-7阻断let-7家族的加工成熟。Lin28对let-7靶基因mRNA具有间接调控作用,富集的mRNA主要在生长进程、信号传导、蛋白质加工及遗传信息表达翻译上发挥作用,与细胞增殖联系紧密。

关键词: Lin28, let-7家族, 人牙髓干细胞, 慢病毒载体转染, IGF2BPs

Abstract: PURPOSE: Bioinformatics was used to analyze the possible mechanism of RNA binding protein Lin28 regulating proliferation of human dental pulp stem cells (hDPSCs). METHODS: Lentivirus-mediated stable transfection of Lin28 gene into hDPSCs was performed, and RNA binding protein immunoprecipitation was used to detect the concentration and total amount of mRNA enriched with overexpression of Lin28. The changes of Lin28-enriched mRNA and related pathways were detected by RIP sequencing. The RIP-seq sequencing results were verified by RIP-PCR. The miRNA expression levels of Lin28 and let-7 family members were detected by real-time PCR. TargetScan (http://www.targetscan.org/vert_71/) was used to predict the let-7a relevant target gene mRNA and specific binding sites. Western blot was used to detect the expression levels of let-7a related target genes IGF1R, IRS2, and IGF2BPs family proteins. RESULTS: RNA binding protein Lin28, as an important mRNA regulatory factor, played a key role in cell growth and development, tissue metabolism, etc. After Lin28 enrichment expression, pri-let-7a/b/c/d/e/f/i/98 and pre-let-7a/b/c/d/e/f/i/98 decreased to different extents. Overexpression of Lin28 could up-regulate the expression levels of let-7-related target genes IGF2BP1 and IGF2BP2 proteins. CONCLUSIONS: Lin28 blocks the processing and maturation of let-7 family through specific complementary binding of pri-let-7 and pre-let-7 in multiple pathways. Lin28 has an indirect regulatory effect on the mRNA of let-7 target genes, and the enriched mRNAs mainly play a role in the growth process, signaling, protein processing, and the translation of the expression of genetic information, which is closely associated with cell growth and proliferation.

Key words: Lin28, Let-7 family, Human dental pulp stem cells, Lentiviral vector transfection, IGF2BPs

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