lincRNA-EPS/miR-24-3p/BMP2信号轴对牙釉质发育影响的体外实验研究

doi:10.12439/kqhm.1005-4979.2024.06.003

摘要/Abstract

摘要：

目的：在体外探究长链基因间非编码RNA-EPS（long intergenic non-coding RNA-EPS，lincRNA-EPS）/微小RNA（microRNA，miRNA）-24-3p/骨形态发生蛋白2（bone morphogenetic protein 2，BMP2）信号轴对牙釉质发育的影响。方法：将lincRNA-EPS过表达质粒和miR-24-3p mimics转染至LS8细胞中，并通过实时定量聚合酶链反应（real-time quantitative polymerase chain reaction，RT-qPCR）分别检测其转染效率；应用RT-qPCR分别检测转染后成釉相关基因（AMELX、AMBN、AMTN、MMP20）和BMP2的mRNA相对表达水平；应用蛋白质印迹法（Western blotting）检测转染后BMP2的蛋白表达水平；采用独立样本t检验进行组间定量比较。结果：显微镜下荧光图像、RT-qPCR结果显示，lincRNA-EPS过表达质粒和miR-24-3p mimics均转染成功；lincRNA-EPS过表达质粒转染后成釉相关基因（AMELX、AMBN、AMTN、MMP20）、BMP2的mRNA及蛋白表达均显著升高，miR-24-3p mimics转染后成釉相关基因、BMP2的mRNA及蛋白表达均显著降低，lincRNA-EPS能在一定程度上抑制miR-24-3p对BMP2的调控作用，差异具有统计学意义（P<0.05）。结论：牙釉质发育受lincRNA-EPS/miR-24-3p/BMP2信号轴的调控，lincRNA-EPS可以影响miR-24-3p对下游BMP2的表达抑制，从而促进釉质发育。

关键词: 长链基因间非编码RNA-EPS, 微小RNA-24-3p, 骨形态发生蛋白2, 牙釉质发育

Abstract:

Objective: To investigate the effect of long intergenic non-coding RNA-EPS (lincRNA-EPS)/ microRNA (miRNA)-24-3p/ bone morphogenetic protein 2 (BMP2) signal axis on enamel development in vitro. Methods: The overexpressed plasmid of lincRNA-EPS and miR-24-3p mimics were transfected into LS8 cells and their transfection efficiency was detected by real-time quantitative polymerase chain reaction (RT-qPCR). RT-qPCR was used to detect the relative mRNA expression of amelogenesis-related genes (AMELX, AMBN, AMTN, MMP20) and BMP2 after transfection. Western blotting was used to detect the protein expression of BMP2 after transfection. Independent sample t test was used for quantitative comparison between groups. Results: Fluorescence images under microscope and RT-qPCR results showed that lincRNA-EPS overexpression plasmid and miR-24-3p mimics were transfected successfully. The relative mRNA expression of amelogenesis-related genes and BMP2 as well as the protein expression of BMP2 were significantly increased after transfection with lincRNA-EPS overexpression plasmid, while the relative mRNA expression of amelogenesis-related genes and BMP2 as well as the protein expression of BMP2 were significantly decreased after transfection with miR-24-3p mimics. To a certain extent, lincRNA-EPS can inhibit the regulatory effect of miR-24-3p on BMP2, and the difference was statistically significant (P<0.05). Conclusion: Enamel development is regulated by lincRNA-EPS/miR-24-3p/BMP2 signal axis, and lincRNA-EPS can affect the inhibition of miR-24-3p on downstream BMP2 expression, thus promoting enamel development.

Key words: long intergenic noncoding RNA-EPS, microRNA-24-3p, bone morphogenetic protein 2, enamel development

中图分类号:

R782

俞秀君, 苏俭生. lincRNA-EPS/miR-24-3p/BMP2信号轴对牙釉质发育影响的体外实验研究[J]. 《口腔颌面外科杂志》, 2024, 34(6): 427-434.

YU Xiujun, SU Jiansheng. Effect of lincRNA-EPS/miR-24-3p/BMP2 signal axis on enamel development: An experimental study in vitro[J]. 《Journal of Oral and Maxillofacial Surgery》, 2024, 34(6): 427-434.

https://journal06.magtech.org.cn/Jweb_joms/CN/Y2024/V34/I6/427

图/表 7

表1 引物序列表

Table 1 Primer sequences

基因	正向引物(5'-3')	反向引物(5'-3')
GAPDH	AGGTCGGTGTGAACGGATTTG	TGTAGACCATGTAGTTGAGGTCA
lincRNA-EPS	TCTCGATCTCACTGCATGGC	TCAGCTGTAGGATGGGAGG
BMP2	TTGGACACCAGGTTAGTGAATCA	TCTCCTCTAAATGGGCCACTT
AMELX	CCCCAGTCACCTCTGCATC	GCTGCATGGAGAACAGTGG
AMBN	ATGAAGGGCCTGATCCTGTTC	GTCTCATTGTCTCAAGGCTCAAA
AMTN	ATCAGCCCAGTCATTACCAAAG	AGGTCTGACCCCAGAGTGAG
MMP20	GGCGAGATGGTGGCAAGAG	CTGGGAAGAGGCGGTAGTT
U6	GCTTCGGCAGCACATATACTAAAAT	CGCTTCACGAATTTGCGTGTCAT
miR-24-3p	GCGTGGCTCAGTTCAGCAG	AGTGCAGGGTCCGAGGTATT

图1 lincRNA-EPS和miR-24-3p的过表达 A—F.转染后各组细胞荧光图像（×40，×100）；G.转染后lincRNA-EPS的mRNA相对表达水平；H.转染后miR-24-3p的相对表达水平。***表示P<0.001，与EPS组比较；###表示P<0.001，与miR-NC-mimics组比较。

Figure 1 Overexpression of lincRNA-EPS and miR-24-3p

图2 lincRNA-EPS过表达质粒转染后BMP2表达 A. BMP2的mRNA相对表达水平，**表示P<0.01，与NC组比较；B. BMP2的蛋白表达条带。

Figure 2 Expression of BMP2 after transfection with lincRNA-EPS overexpression plasmid

图3 miR-24-3p mimics转染后BMP2表达 A. BMP2的mRNA相对表达水平，**表示P<0.01，与miR-NC mimics组比较；B. BMP2的蛋白表达条带。

Figure 3 Expression of BMP2 after transfection with miR-24-3p mimics

图4 lincRNA-EPS和miR-24-3p mimics共转染48 h后的BMP2的表达 A.共转染后各组BMP2的mRNA相对表达水平，#表示P<0.05，与NC+miR-24-3p mimics组比较；**表示P<0.01，与EPS+miR-NC mimics组比较；B.共转染后BMP2的蛋白表达条带。

Figure 4 Expression of BMP2 after co-transfection with lincRNA-EPS overexpression plasmid and miR-24-3p mimics for 48 h

图5 转染后NC组和EPS组的釉质发育相关基因mRNA表达水平 A—D.依次为AMELX、AMBN、AMTN、MMP20 mRNA相对表达量。**表示P<0.01，***表示P<0.001，与NC组比较。

Figure 5 mRNA expression levels of amelogenesis-related genes in NC and EPS groups after transfection

图6 转染后miR-NC mimics组和miR-24-3p mimics组的釉质发育相关基因mRNA表达水平 A—D.依次为AMELX、AMBN、AMTN和MMP20基因相对表达量。*表示P<0.05，**表示P<0.01，***表示P<0.001，与miR-NC-mimics组比较。

Figure 6 mRNA expression levels of amelogenesis-related genes in miR-NC mimics and miR-24-3p mimics groups after transfection

参考文献 23

[1]	Zhang H, Gong XY, Xu XQ, et al. Tooth number abnormality: From bench to bedside[J]. Int J Oral Sci, 2023, 15(1): 5. doi: 10.1038/s41368-022-00208-x pmid: 36604408
[2]	Zhang YD, Chen Z, Song YQ, et al. Making a tooth: Growth factors, transcription factors, and stem cells[J]. Cell Res, 2005, 15(5): 301-316. doi: 10.1038/sj.cr.7290299 pmid: 15916718
[3]	Graf D, Malik Z, Hayano S, et al. Common mechanisms in development and disease: BMP signaling in craniofacial development[J]. Cytokine Growth Factor Rev, 2016, 27: 129-139.
[4]	Yuan GH, Yang GB, Zheng YQ, et al. The non-canonical BMP and Wnt/β-catenin signaling pathways orchestrate early tooth development[J]. Development, 2015, 142(1): 128-139. doi: 10.1242/dev.117887 pmid: 25428587
[5]	Liu CW, Guo H, Shi C, et al. BMP signaling in the development and regeneration of tooth roots: From mechanisms to applications[J]. Front Cell Dev Biol, 2023, 11: 1272201.
[6]	Liu MM, Goldman G, MacDougall M, et al. BMP signaling pathway in dentin development and diseases[J]. Cells, 2022, 11(14): 2216.
[7]	Wang Y, Li L, Zheng Y, et al. BMP activity is required for tooth development from the Lamina to bud stage[J]. J Dent Res, 2012, 91(7): 690-695. doi: 10.1177/0022034512448660 pmid: 22592126
[8]	Gao ZH, Wang LX, Wang F, et al. Expression of BMP2/4/7 during the odontogenesis of deciduous molars in miniature pig embryos[J]. J Mol Histol, 2018, 49(5): 545-553. doi: 10.1007/s10735-018-9792-1 pmid: 30099666
[9]	Malik Z, Alexiou M, Hallgrimsson B, et al. Bone morphogenetic protein 2 coordinates early tooth mineralization[J]. J Dent Res, 2018, 97(7): 835-843. doi: 10.1177/0022034518758044 pmid: 29489425
[10]	Liu J, Saiyin W, Xie XH, et al. Ablation of Fam20c causes amelogenesis imperfecta via inhibiting Smad dependent BMP signaling pathway[J]. Biol Direct, 2020, 15(1): 16. doi: 10.1186/s13062-020-00270-7 pmid: 33028367
[11]	Mohr AM, Mott JL. Overview of microRNA biology[J]. Semin Liver Dis, 2015, 35(1): 3-11. doi: 10.1055/s-0034-1397344 pmid: 25632930
[12]	Sehic A, Tulek A, Khuu C, et al. Regulatory roles of microRNAs in human dental tissues[J]. Gene, 2017, 596: 9-18. doi: S0378-1119(16)30804-6 pmid: 27725267
[13]	Jin Y, Wang CL, Cheng S, et al. MicroRNA control of tooth formation and eruption[J]. Arch Oral Biol, 2017, 73: 302-310. doi: S0003-9969(16)30223-0 pmid: 27835837
[14]	Michon F, Tummers M, Kyyrönen M, et al. Tooth morphogenesis and ameloblast differentiation are regulated by micro-RNAs[J]. Dev Biol, 2010, 340(2): 355-368. doi: 10.1016/j.ydbio.2010.01.019 pmid: 20102707
[15]	Oommen S, Otsuka-Tanaka Y, Imam N, et al. Distinct roles of microRNAs in epithelium and mesenchyme during tooth development[J]. Dev Dyn, 2012, 241(9): 1465-1472.
[16]	Jevnaker AM, Osmundsen H. MicroRNA expression profiling of the developing murine molar tooth germ and the developing murine submandibular salivary gland[J]. Arch Oral Biol, 2008, 53(7): 629-645.
[17]	Cao HJ, Jheon A, Li X, et al. The Pitx2: miR-200c/141: Noggin pathway regulates Bmp signaling and ameloblast differentiation[J]. Development, 2013, 140(16): 3348-3359.
[18]	Chan JJ, Tay Y. Noncoding RNA: RNA regulatory networks in cancer[J]. Int J Mol Sci, 2018, 19(5): 1310.
[19]	郝晨笛, 苏俭生. 长链非编码RNA-EPS对小鼠牙釉质发育的影响[J]. 口腔颌面外科杂志, 2021, 31(5): 272-277. doi: 10.3969/j.issn.1005-4979.2021.05.002
[20]	Dhanoa JK, Sethi RS, Verma R, et al. Long non-coding RNA: Its evolutionary relics and biological implications in mammals: A review[J]. J Anim Sci Technol, 2018, 60: 25.
[21]	Bridges MC, Daulagala AC, Kourtidis A. LNCcation: lncRNA localization and function[J]. 2021, 220(2): e202009045.
[22]	Chen LL, Song Z, Wu JY, et al. LncRNA DANCR sponges miR-216a to inhibit odontoblast differentiation through upregulating c-Cbl[J]. Exp Cell Res, 2020, 387(1): 111751.
[23]	Zhong JL, Tu XR, Kong YY, et al. LncRNA H19 promotes odontoblastic differentiation of human dental pulp stem cells by regulating miR-140-5p and BMP-2/FGF9[J]. Stem Cell Res Ther, 2020, 11(1): 202. doi: 10.1186/s13287-020-01698-4 pmid: 32460893

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