Abstract
Dental fluorosis is a common chemical disease. It is currently unclear how fluorosis occurs at the molecular level. We used miRNA-seq to look at the differences between miRNAs in the cell line of ameloblasts LS8 that had been treated with 3.2 mmol/L NaF. We also performed gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. miR-1a-3p levels were significantly lower in mouse LS8 cells treated with 3.2 mmol/L NaF, and miR-1a-3p-targeted genes were significantly enriched in the MAPK pathway. LS8 cells were divided into four groups: control, NaF, NaF+miR-1a-3p mimics, and NaF+miR-1a-3p mimics normal control groups. Cellular morphology was observed by an inverted microscope, and the proliferation activity of LS8 cells was assessed by Cell Counting Kit-8 (CCK-8). Using the real-time quantitative polymerase chain reaction (RT-qPCR), transcription levels of miR-1a-3p and Map3k1 were detected. The expressions of Bax, Bcl-2, Map3k1, p38MAPK, ERK1/2, p-p38MAPK, and p-ERK1/2 were measured by Western blot. After bioinformatics analysis, we used a luciferase reporter assay (LRA) to validate the target of miR-1a-3p, showing that miR-1a-3p could inhibit apoptosis while increasing proliferation in fluoride-exposed LS8 cells. Generally, miR-1a-3p might directly inhibit Map3k1, reduce MAPK signal pathway activation, and promote phosphorylation. Thus, our findings revealed that the interaction of miR-1a-3p with its target gene Map3k1 and MAPK signal pathway might decrease the apoptosis of LS8 cells treated with 3.2 mmol/L NaF.
• Original abstract online at https://link.springer.com/article/10.1007/s12011-023-03869-9
Excerpt:
Introduction
Endemic fluorosis is a chemical disease that affects people all over the world. It is also one of the most serious diseases that naturally occur in China. Dental fluorosis is a precursor and specific indicator of chronic fluorosis in the oral cavity and has a significant impact around the world. When dental fluorosis develops, it not only changes how a person looks but can also affect their mental health in different ways. According to the results of the fourth oral epidemiological survey, 13.4% of 12-year-olds in China have dental fluorosis [1]. It is currently unclear why tooth fluorosis occurs, and its molecular mechanisms are not completely clear. Since the abnormal posttranscriptional cascades causing bone and tooth deformities are unknown, there is no prevention and treatment for dental fluorosis. LS8 cells are the key cells in the development of enamel. Many studies showed that excessive fluoride can damage LS8 cells and cause dental fluorosis [2, 3]. The MAPK pathway is an important pathway that affects the formation of enamel, matrix secretion, and cusp formation [4]. Odontoblast differentiation is also affected by the MAPK signal pathway [5]. Fluoride can mediate the expressions of downstream genes through the MAPK signal pathway to participate in dental fluorosis development [6,7,8].
microRNA (miRNA) is an endogenous (18–25 nt) nonprotein coding RNA molecule that regulates posttranscriptional gene expression by pairing with 3’UTR of the target mRNA. Many pathophysiological processes are regulated by miRNAs [9]. Studies demonstrated that miRNAs play an important role in enamel development by regulating epithelial cell differentiation, enamel mineralization, and extracellular matrix receptor interaction. In recent years, epigenetic studies on fluorosis have emerged. Some studies reported that miRNAs affect epigenetic modification by regulating target genes, and miRNAs and fluoride are closely related. Excessive fluoride exposure may significantly disrupt the expression pattern of miRNA [10,11,12]. Thus far, the important role of miRNA in fluorosis and tooth development has been widely recognized. Therefore, our team thought that ameloblast apoptosis caused by excessive fluoride might involve changes in miRNA expression. The experimental results showed that excessive sodium fluoride promoted apoptosis of LS8 cells and inhibited their proliferation, and the effect increased with concentration and time. In this study, we analyzed previous sequencing data to find differentially expressed miRNAs of LS8 cells undergoing apoptosis and treated with 3.2 mmol/L NaF and explore the mechanism of regulating fluoride-stained apoptosis of LS8 cells.
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