基于转录组测序探究氟对成釉细胞影响的分子机制

doi:10.13701/j.cnki.kqyxyj.2025.05.011

口腔医学研究 ›› 2025, Vol. 41 ›› Issue (5): 420-425.DOI: 10.13701/j.cnki.kqyxyj.2025.05.011

基于转录组测序探究氟对成釉细胞影响的分子机制

姚姝冉^1,2, 翁清清^1,2, 朱忆颖^1,2, 刘佳^1,2, 张颖^1,2*

1.上海市口腔医院上海 201103;
2.上海市颅颌面发育与疾病重点实验室上海 201103

收稿日期:2024-10-21 出版日期:2025-05-28 发布日期:2025-05-26
通讯作者: ^*张颖,E-mail:zhangyingcmu@vip.163.com
作者简介:姚姝冉(1997~ ),女,江苏连云港人,硕士,医师,研究方向:氟牙症致病机制研究。
基金资助:
国家自然科学基金(编号:81773369)上海市自然科学基金 (编号:16ZR1430600)

Exploring Molecular Mechanisms of Fluoride's Impact on Ameloblasts Based on Transcriptome Sequencing

YAO Shuran^1,2, WENG Qingqing^1,2, ZHU Yiying^1,2, LIU Jia^1,2, ZHANG Ying^1,2*

1. Shanghai Stomatological Hospital, Shanghai 201103, China;
2. Shanghai Key Laboratory of Craniomaxillofacial Development and Diseases, Shanghai 201103, China

Received:2024-10-21 Online:2025-05-28 Published:2025-05-26

摘要/Abstract

摘要： 目的: 基于转录组测序(transcriptome sequencing, RNA-seq)分析氟化钠(NaF)对小鼠成釉细胞系LS8细胞基因表达的影响,分析关键差异表达基因(differentially expressed genes, DEGs),为探究氟牙症发生的致病机制提供新思路。方法: 实验分为两组,空白对照组和NaF组(1.5 mmol/L)处理LS8细胞24 h后收集样本,进行RNA-seq分析,筛选出关键DEGs,采用京都基因与基因组百科全书(Kyoto encyclopedia of genes and genomes, KEGG)对基因进行通路富集分析。通过实时荧光定量聚合酶链反应技术(real-time quantitative PCR, RT-qPCR)验证测序结果的准确性。此外,通过Western blot验证相关信号通路蛋白的表达情况。结果: NaF处理24 h后,RNA-seq共检测筛选出 DEGs共104个,其中上调基因34个,下调基因70个。KEGG富集分析结果显示,磷脂酰肌醇3-激酶/蛋白激酶B (phosphatidylinositol 3-kinase/protein kinase B, PI3K/Akt)和丝裂原活化蛋白激酶(mitogen-activated protein kinase, MAPK)等信号通路在NaF处理后的成釉细胞中显著富集。RT-qPCR发现PI3K、Akt、MAPK mRNA表达下降(P<0.05),与RNA-seq结果一致。Western blot结果显示p-PI3K/PI3K、p-Akt/Akt、p-p44/p42MAPK/ p44/p42MAPK蛋白相对表达量降低(P<0.05)。结论: PI3K/Akt、MAPK信号通路可能参与氟对成釉细胞毒性作用的调控过程。

关键词: 转录组测序, 氟牙症, 成釉细胞, PI3K/Akt信号通路, 丝裂原活化蛋白激酶信号通路

Abstract: Objective: To investigate the impact of sodium fluoride (NaF) on gene expression in the mouse ameloblast cell line LS8, and to identify differentially expressed genes (DEGs) utilizing transcriptome sequencing (RNA-seq). Methods: The research was divided into two groups: the control group receiving no treatment and the experimental group exposed to 1.5 mmol/L NaF for 24 hours. Post-treatment, samples were collected for RNA-seq analysis. The DEGs were identified, and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was conducted. To validate the accuracy of the sequencing results, real-time quantitative PCR (RT-qPCR) was employed, while Western blotting confirmed the expression levels of relevant signaling pathway proteins. Results: After 24 hours of NaF exposure, RNA-seq detected 104 DEGs-34 up-regulated and 70 down-regulated. KEGG enrichment analysis indicated notable activation of the phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) and mitogen-activated protein kinase (MAPK) signaling pathways in LS8 cells. RT-qPCR showed decreased mRNA levels for PI3K, Akt, and MAPK (P<0.05), corroborating findings from RNA-seq analyses. Furthermore, Western blot results revealed lower relative expression levels of p-PI3K/PI3K, p-Akt/Akt, and p-p44/p42MAPK/p44/p42MAPK (P<0.05). Conclusion: Both PI3K/Akt and MAPK signaling pathways may play crucial roles in mediating the toxic effects of fluoride on ameloblasts.

Key words: transcriptome sequencing, dental fluorosis, ameloblasts, PI3K/Akt signaling pathway, MAPK signaling pathway

姚姝冉, 翁清清, 朱忆颖, 刘佳, 张颖. 基于转录组测序探究氟对成釉细胞影响的分子机制[J]. 口腔医学研究, 2025, 41(5): 420-425.

YAO Shuran, WENG Qingqing, ZHU Yiying, LIU Jia, ZHANG Ying. Exploring Molecular Mechanisms of Fluoride's Impact on Ameloblasts Based on Transcriptome Sequencing[J]. Journal of Oral Science Research, 2025, 41(5): 420-425.

参考文献

[1] Wei W, Pang S, Sun D. The pathogenesis of endemic fluorosis: Research progress in the last 5 years [J]. J Cell Mol Med, 2019, 23(4):2333-2342.
[2] Revelo-Mejia IA, Hardisson A, Rubio C, et al. Dental fluorosis: the risk of misdiagnosis-a review [J]. Biol Trace Elem Res, 2021, 199(5):1762-1770.
[3] Suzuki M, Bandoski C, Bartlett JD. Fluoride induces oxidative damage and SIRT1/autophagy through ROS-mediated JNK signaling [J]. Free Radic Biol Med, 2015, 89:369-378.
[4] Yang S, Song D, Wang R, et al. Sodium fluoride-induced autophagy of ameloblast-like cells via the p-ULk1/ATG13/LC3B pathway in vitro [J]. Oral Dis, 2024, 30(7):4518-4527.
[5] Yao S, Weng Q, Zhu Y, et al. Excessive fluoride impairs autophagy flux in ameloblasts which is prevented by the autophagy activator rapamycin [J]. Environ Toxicol, 2023, 38(1):193-204.
[6] Lv H, Yang H, Wang Z, et al. Nrf2 signaling and autophagy are complementary in protecting lipopolysaccharide/d-galactosamine-induced acute liver injury by licochalcone A [J]. Cell Death Dis, 2019, 10(4):313.
[7] Dou Z, Pan JA, Dbouk HA, et al. Class IA PI3K p110beta subunit promotes autophagy through Rab5 small GTPase in response to growth factor limitation [J]. Mol Cell, 2013, 50(1):29-42.
[8] El-Shoura E, Abdelzaher LA, Mahmoud NI, et al. Combined sulforaphane and beta-sitosterol mitigate olanzapine-induced metabolic disorders in rats: Insights on FOXO, PI3K/AKT, JAK/STAT3, and MAPK signaling pathways [J]. Int Immunopharmacol, 2024, 140:112904.
[9] Mokhfi FZ, Al AM, Zehravi M, et al. Alkaloid-based modulators of the PI3K/Akt/mTOR pathway for cancer therapy: Understandings from pharmacological point of view [J]. Chem Biol Interact, 2024, 402:111218.
[10] Huang T, Zhang C, Shang Z, et al. Bone mesenchymal stem cells improve cholestatic liver fibrosis by targeting ULK1 to regulate autophagy through PI3K/AKT/mTOR pathway [J]. Stem Cells Transl Med, 2024, 13(7):648-660.
[11] Cheng L, Xiang S, Yu Q, et al. Paeoniflorin inhibits PRAS40 interaction with Raptor to activate mTORC1 to reverse excessive autophagy in airway epithelial cells for asthma [J]. Phytomedicine, 2024, 134:155946.
[12] Korkmaz R, Yuksek V, Dede S. The Effects of sodium fluoride (NaF) treatment on the PI3K/Akt signal pathway in NRK-52E cells [J]. Biol Trace Elem Res, 2022, 200(7):3294-3302.
[13] Linghu Y, Deng CN, He L, et al. Fluoride induces osteoblast autophagy by inhibiting the PI3K/AKT/mTOR signaling pathway in vivo and in vitro [J]. Exp Biol Med (Maywood), 2023, 248(13):1159-1172.
[14] Kim JH, Hong SK, Wu PK, et al. Raf/MEK/ERK can regulate cellular levels of LC3B and SQSTM1/p62 at expression levels [J]. Exp Cell Res, 2014, 327(2):340-352.
[15] Liu YL, Lai F, Wilmott JS, et al. Noxa upregulation by oncogenic activation of MEK/ERK through CREB promotes autophagy in human melanoma cells [J]. Oncotarget, 2014, 5(22):11237-11251.
[16] Sivaprasad U, Basu A. Inhibition of ERK attenuates autophagy and potentiates tumour necrosis factor-alpha-induced cell death in MCF-7 cells [J]. J Cell Mol Med, 2008, 12(4):1265-1271.
[17] Cui D, Xiao J, Zhou Y, et al. Epiregulin enhances odontoblastic differentiation of dental pulp stem cells via activating MAPK signalling pathway [J]. Cell Prolif, 2019, 52(6): e12680.
[18] Bai Y, Cheng X, Liu X, et al. Transforming growth factor-beta1 promotes early odontoblastic differentiation of dental pulp stem cells via activating AKT, Erk1/2 and p38 MAPK pathways [J]. J Dent Sci, 2023, 18(1):87-94.
[19] Greenblatt MB, Kim JM, Oh H, et al. p38alpha MAPK is required for tooth morphogenesis and enamel secretion [J]. J Biol Chem, 2015, 290(1):284-295.
[20] Jia J, Yang F, Yang M, et al. P38/JNK signaling pathway mediates the fluoride-induced down-regulation of Fam83h [J]. Biochem Biophys Res Commun, 2016, 471(3):386-390.
[21] Zhao L, Su J, Liu S, et al. MAP kinase phosphatase MKP-1 regulates p-ERK1/2 signaling pathway with fluoride treatment [J]. Biochem Biophys Res Commun, 2021, 542:65-72.
[22] Chen T, Gu Y, Bai GH, et al. MiR-1a-3p inhibits apoptosis in fluoride-exposed LS8 cells by targeting Map3k1 [J]. Biol Trace Elem Res, 2024, 202(6):2720-2729.
[23] 张颖,马林,李健,等.过量氟诱导成釉细胞钙超载及细胞凋亡的实验研究[J].华西口腔医学杂志,2014,32(6):542-546.
[24] 李玲,刘怡,彭睿,等.氟对成釉细胞钙稳态的影响及其机制的研究进展[J].中国地方病防治杂志,2016,31(1):34-36.
[25] Chen R, Zhao LD, Liu H, et al. Fluoride induces neuroinflammation and alters Wnt signaling pathway in BV2 microglial cells [J]. Inflammation, 2017, 40(4):1123-1130.

基于转录组测序探究氟对成釉细胞影响的分子机制

Exploring Molecular Mechanisms of Fluoride's Impact on Ameloblasts Based on Transcriptome Sequencing

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