中国组织工程研究

中国组织工程研究 ›› 2024, Vol. 28 ›› Issue (19): 3003-3011.doi: 10.12307/2024.151

• 干细胞培养与分化 stem cell culture and differentiation • 上一篇    下一篇

miR-135a-5p靶向抑制Kif3B调控小鼠腭胚突间充质细胞的自噬

冯文宣1，廉舒博1，王  哲1，陈  靖1，何  苇1，2   

  1. 1遵义医科大学，口腔医学院，贵州省遵义市   563000；2遵义医科大学附属口腔医院，贵州省遵义市   563000
  • 收稿日期:2023-04-15 接受日期:2023-05-15 出版日期:2024-07-08 发布日期:2023-09-26
  • 通讯作者: 何苇，博士，副教授，遵义医科大学，口腔医学院，贵州省遵义市 563000；遵义医科大学附属口腔医院，贵州省遵义市 563000
  • 作者简介:冯文宣，男，1996年生，河南省南阳市人，汉族，遵义医科大学在读硕士，主要从事口腔颌面部发育与畸形的研究。
  • 基金资助:
    国家自然科学基金(82160176)，项目负责人：何苇；遵义医科大学研究生科研基金立项项目(ZYK65)，项目负责人：冯文宣

miR-135a-5p regulates autophagy of mouse embryonic palatal mesenchymal cells via targeting Kif3B

Feng Wenxuan1, Lian Shubo1, Wang Zhe1, Chen Jing1, He Wei1, 2   

  1. 1School of Stomatology, Zunyi Medical University, Zunyi 563000, Guizhou Province, China; 2Hospital of Stomatology, Zunyi Medical University, Zunyi 563000, Guizhou Province, China
  • Received:2023-04-15 Accepted:2023-05-15 Online:2024-07-08 Published:2023-09-26
  • Contact: He Wei, MD, Associate professor, School of Stomatology, Zunyi Medical University, Zunyi 563000, Guizhou Province, China; Hospital of Stomatology, Zunyi Medical University, Zunyi 563000, Guizhou Province, China
  • About author:Feng Wenxuan, Master candidate, School of Stomatology, Zunyi Medical University, Zunyi 563000, Guizhou Province, China
  • Supported by:
    National Natural Science Foundation of China, No. 82160176 (to HW); Postgraduate Research Fund Project of Zunyi Medical University, No. ZYK65 (to FWX)

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摘要：


文题释义：

microRNAs：是一种非编码短链RNA(大约22个核苷酸)，通过与靶点mRNA的3’-UTR相结合，发挥转录后水平调控作用。
细胞自噬：是在自噬相关基因的作用下，真核细胞通过溶酶体对损伤细胞器及胞浆中蛋白质进行降解的一种细胞行为。在正常生理条件下，细胞自噬处于较低水平以调控细胞的稳态，如蛋白质或细胞器的更新。


背景：研究表明，在地塞米松诱导腭裂的小鼠胚胎腭突间充质细胞中miR-135a-5p呈高表达，初级纤毛及其介导的Shh信号通路参与小鼠胚胎腭突间充质细胞的自噬。由此猜测miR-135a-5p可能通过初级纤毛及其介导的Shh信号途径调控小鼠胚胎腭突间充质细胞的自噬。

目的：探讨miR-135a-5p对小鼠胚胎腭突间充质细胞自噬的调控作用。
方法：体外提取并培养C57BL/6J小鼠胚胎腭突间充质细胞。细胞转染分别设置为：①对照组、miR-135a-5p阴性对照组、miR-135a-5p模拟物组。②NC+miR-NC组、KIF3B过表达组、miR-135a-5p+KIF3B组。qRT-PCR验证miR-135a-5p、KIF3B的转染效率；透射电镜观察各组细胞中自噬小体/自噬溶酶体的数目；免疫荧光技术测定自噬标记物LC3B的荧光表达程度；Western blot检测KIF3B、LC3和P62的蛋白表达。③miR-135a-5p阴性对照组、SAG处理组、SAG+miR-135a-5p组。qRT-PCR检测Shh信号下游关键转录因子Gli3的mRNA表达水平；Western blot检测自噬相关蛋白LC3和P62的蛋白表达。

结果与结论：①过表达miR-135a-5p后，细胞内自噬小体/自噬溶酶体数量显著增多(P < 0.01)；LC3B的荧光密度显著升高(P < 0.01)，KIF3B、P62的蛋白表达降低(P < 0.01)，LC3的蛋白表达升高；②过表达KIF3B后，细胞内自噬小体/自噬溶酶体数目明显减少(P < 0.01)，LC3B的荧光密度降低(P < 0.01)，P62的蛋白表达升高(P < 0.01)，LC3的蛋白表达下降(P < 0.01)；miR-135a-5p靶向抑制KIF3B的表达(P < 0.01)，并使自噬小体/自噬溶酶体数目、LC3B的荧光强度以及LC3的蛋白表达得到回升(P < 0.01)，P62的蛋白表达下降(P < 0.01)；③SAG使Gli3的mRNA表达明显升高(P < 0.01)，P62的蛋白表达升高(P < 0.01)，LC3的蛋白表达下降(P < 0.01)；加入miR-135a-5p后，Gli3的mRNA表达显著下降(P < 0.01)，P62的蛋白表达下降(P < 0.01)，LC3的蛋白表达回升(P < 0.01)；④结果表明：miR-135a-5p靶向抑制KIF3B并且可能通过负向调控Shh信号通路促进小鼠胚胎间充质细胞自噬。

https://orcid.org/0000-0002-7483-8143 (冯文宣) 

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

关键词: 自噬, 胚胎腭突间充质细胞, miRNA, KIF3B, Shh信号通路, 腭裂

Abstract: BACKGROUND: Studies demonstrated that miR-135a-5p was highly expressed in mouse embryonic palatal mesenchymal cells with cleft palate induced by dexamethasone. The primary cilium and its mediated Shh signaling pathway were involved in the autophagy of mouse embryonic palatal mesenchymal cells. It is speculated that miR-135a-5p may regulate autophagy in mouse embryonic palatal mesenchymal cells through primary cilia and its mediated Shh signaling pathway.
OBJECTIVE: To investigate the regulatory effect of miR-135a-5p on autophagy of mouse embryonic palatal mesenchymal cells.
METHODS: In vitro, palatal mesenchymal cells from C57BL/6J mouse embryos were extracted and cultured. Cell transfections were set up as follows: (1) the cells were divided into control group, miR-135a-5p negative control group and miR-135a-5p mimic group; (2) NC+miR-NC group, KIF3B overexpression group, and miR-135a-5p+KIF3B group: qRT-PCR was performed to verify transfection efficiency of miR-135a-5p and KIF3B. A transmission electron microscope was used to observe the number of autophagosome/autophagolysosome in the cells of each group. The degree of fluorescence expression of autophagy marker LC3B was determined by the immunofluorescence technique. The protein expression of KIF3B, LC3 and P62 was determined by western blot assay. (3) The cells were divided into miR-135a-5p negative control group, and SAG treated group, and SAG+miR-135a-5p group. qRT-PCR was used to detect the mRNA expression levels of Gli3, a key transcription factor downstream of Shh signaling. The protein expressions of autophagy-related proteins LC3 and P62 were detected by western blot assay. 
RESULTS AND CONCLUSION: (1) After overexpression of miR-135a-5p, the number of autophagosome/autophagolysosome was significantly increased (P < 0.01). The fluorescence density of LC3B increased significantly (P < 0.01); the protein expression of KIF3B and P62 decreased (P < 0.01), and the protein expression of LC3 increased. (2) After overexpression of KIF3B, the number of autophagosome/autophagolysosome was significantly decreased (P < 0.01); the fluorescence density of LC3B was decreased (P < 0.01); the protein expression of P62 was increased (P < 0.01), and the protein expression of LC3 was decreased (P < 0.01). Targeted expression of KIF3B was inhibited by miR-135a-5p (P < 0.01); the number of autophagosome/autophagolysosome, the fluorescence intensity of LC3B as well as the protein expression of LC3 were reversed (P < 0.01) and the protein expression of P62 was decreased (P < 0.01). (3) SAG significantly increased the mRNA expression of Gli3 (P < 0.01), increased the protein expression of P62 (P < 0.01), and decreased the protein expression of LC3 (P < 0.01). When miR-135a-5p was added, Gli3 mRNA expression was significantly decreased (P < 0.01); P62 protein expression was decreased (P < 0.01), and LC3 protein expression was reversed (P < 0.01). (4) These results indicate that miR-135a-5p targets the inhibition of KIF3B and promotes autophagy in mouse embryonic mesenchymal cells possibly by negatively regulating the Shh signaling pathway.

Key words: autophagy, embryonic palatal mesenchymal cell, miRNA, KIF3B, Shh signaling pathway, cleft palate

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