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Fluoride resistance capacity in mammalian cells involves complex global gene expression changes

Ran, Shujun, Sun, Ning, Liu, Yun, Zhang, Wu, Li, Yiming, Wei, Limin, Wang, Jia, Liu, Bin·2017·FEBS Open Bio
DOI 10.1002/2211-5463.12236PMC PMC5494298
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Abstract

Fluorine is a bone‐seeking element ubiquitously present in the environment and widely used in many oral hygiene products. In humans, excessive intake of fluoride may cause dental and skeletal fluorosis. However, endemic fluorosis does not appear to develop in a proportion of individuals exposed to the same levels of fluoride. The mechanisms by which mammalian cells resist fluoride are still unclear. In this study, we developed strains of mouse L‐929 cells resistant to different levels of fluoride. High‐throughput RNA‐sequencing analyses of the fluoride‐resistant L‐929 cells indicated that massive changes in global gene expression occurred, compared with the wild‐type L‐929 cells. The main biological processes and functions changed were associated with the extracellular region and matrix, response to stress, receptor binding, and signal transduction. This indicated that high doses of fluoride not only exerted stress on L‐929 cells but also induced functional pathways that helped them adapt to the presence of fluoride or to expel it. These data should prove useful in identifying cellular processes or transporters/channels that play central roles in adaptation to or expulsion of fluoride in humans.

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Figure 1 Inducement of FR strains of L‐929 cells. (A) Inhibition rate of WT L‐929 cells in varying concentrations of fluoride media. WT L929 cells were cultured in media with various concentrations of fluoride. MTT assay was performed according to scheduled date and the OD at the wavelength of 570 nm were obtained by a microplate reader (Bio‐Rad Benchmark, Hercules, CA, USA). (B) Proliferation curves of WT and induced FR strains of L‐929 cells. WT L‐929 cells were cultured in fluoride‐free media while FR strains were cultivated in fluoride media. MTT test was performed according to scheduled date and the 570 nm OD were documented. (C) WT and induced FR strains of L‐929 cells.
Figure 1 Inducement of FR strains of L‐929 cells. (A) Inhibition rate of WT L‐929 cells in varying concentrations of fluoride media. WT L929 cells were cultured in media with various concentrations of fluoride. MTT assay was performed according to scheduled date and the OD at the wavelength of 570 nm were obtained by a microplate reader (Bio‐Rad Benchmark, Hercules, CA, USA). (B) Proliferation curves of WT and induced FR strains of L‐929 cells. WT L‐929 cells were cultured in fluoride‐free media while FR strains were cultivated in fluoride media. MTT test was performed according to scheduled date and the 570 nm OD were documented. (C) WT and induced FR strains of L‐929 cells.CC BY · PubMed Central
Figure 2 Analyses of global gene expression pattern of the fluoride resistance L‐929 cells by RNA‐Seq. (A) Global gene expression correlation study of the WT and 30 p.p.m. FR L‐929 cells. (B) Fold changes of gene expression levels between the WT and 30 p.p.m. FR L‐929 cells. Each dot represents expression levels of a single coding gene. (C) Heat map of the genes with greater than 2‐fold changes in 30 p.p.m. FR L‐929 cells compared to those of the WT L‐929 cells.
Figure 2 Analyses of global gene expression pattern of the fluoride resistance L‐929 cells by RNA‐Seq. (A) Global gene expression correlation study of the WT and 30 p.p.m. FR L‐929 cells. (B) Fold changes of gene expression levels between the WT and 30 p.p.m. FR L‐929 cells. Each dot represents expression levels of a single coding gene. (C) Heat map of the genes with greater than 2‐fold changes in 30 p.p.m. FR L‐929 cells compared to those of the WT L‐929 cells.CC BY · PubMed Central
Figure 3 Biological process analyses of the RNA‐seq results. (A) Major biological processes the 385 changed genes were involved. (B) Ranking of top 10 biological processes by P values. (C) Deeper level 4 analyses of the biological processes the 385 changed genes were involved.
Figure 3 Biological process analyses of the RNA‐seq results. (A) Major biological processes the 385 changed genes were involved. (B) Ranking of top 10 biological processes by P values. (C) Deeper level 4 analyses of the biological processes the 385 changed genes were involved.CC BY · PubMed Central
Figure 4 Cellular component analyses of the RNA‐seq results. (A) Major cellular components the 385 changed genes belong to. (B) Ranking of top 10 cellular components by P values. (C) Deeper level 4 analyses of the cellular component the 385 changed genes belong to.
Figure 4 Cellular component analyses of the RNA‐seq results. (A) Major cellular components the 385 changed genes belong to. (B) Ranking of top 10 cellular components by P values. (C) Deeper level 4 analyses of the cellular component the 385 changed genes belong to.CC BY · PubMed Central
Figure 5 Molecular function analyses of the RNA‐seq results. (A) Major molecular functions the 385 changed genes were involved. (B) Ranking of top 10 molecular functions by P values. (C) Deeper level 4 analyses of the molecular functions the 385 changed genes were involved.
Figure 5 Molecular function analyses of the RNA‐seq results. (A) Major molecular functions the 385 changed genes were involved. (B) Ranking of top 10 molecular functions by P values. (C) Deeper level 4 analyses of the molecular functions the 385 changed genes were involved.CC BY · PubMed Central
Figure 6 KEGG pathway analyses of RNA‐seq results. (A) The main KEGG pathways that the 385 changed genes lye in. (B) Ranking of top 10 pathways by P values. (C) Network analysis of the main signaling pathways that changed in the 30 p.p.m. FR L‐929 cells.
Figure 6 KEGG pathway analyses of RNA‐seq results. (A) The main KEGG pathways that the 385 changed genes lye in. (B) Ranking of top 10 pathways by P values. (C) Network analysis of the main signaling pathways that changed in the 30 p.p.m. FR L‐929 cells.CC BY · PubMed Central
Figure 7 Validation of the key genes that were up‐ or down‐regulated in the 30 p.p.m. FR L‐929 cells. (A) List of the key genes that appear most frequently in the gene ontology analyses. (B) Validation of expression levels of the genes list in (A) by quantitative PCR. Data were represented as mean ± SD, *P < 0.05.
Figure 7 Validation of the key genes that were up‐ or down‐regulated in the 30 p.p.m. FR L‐929 cells. (A) List of the key genes that appear most frequently in the gene ontology analyses. (B) Validation of expression levels of the genes list in (A) by quantitative PCR. Data were represented as mean ± SD, *P < 0.05.CC BY · PubMed Central

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Metadata

Authors
Ran, S., Sun, N., Liu, Y., Zhang, W., Li, Y., Wei, L., Wang, J., & Liu, B.
Affiliations
  • Department of Endodontics and Operative Dentistry Ninth People's Hospital Shanghai Jiao Tong University School of Medicine Shanghai Key Laboratory of Stomatology Shanghai China
  • Department of Physiology and Pathophysiology Fudan University Shanghai China
  • The Ministry of Education Key Laboratory of Metabolism and Molecular Medicine Department of Biochemistry and Molecular Biology School of Basic Medical Sciences Fudan University Shanghai China
  • Center for Dental Research Loma Linda University School of Dentistry CA USA
Venue
FEBS Open Bio
Publisher
Wiley
Published
2017-06-05
Volume
7
Issue
7
Pages
968-980
ISSN
2211-5463
DOI
10.1002/2211-5463.12236
PMC
PMC5494298
Cited by
10
References
0
Language
en
Licence
creativecommons.org/licenses/by/4.0/ (vor)
Funders
National Natural Science Foundation of China (81100753)

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