Transcriptomic analysis of MicroRNA expression in enamel-producing cells.

There is little evidence for the involvement of microRNAs (miRNAs) in the regulation of circadian rhythms during enamel development. Few studies have used ameloblast-like cell line LS8 to study the circadian rhythm of gene activities related to enamel formation. However, the transcriptomic analysis of miRNA expression in LS8 cells has not been established yet. In this study, we analyze the oscillations of miRNAs in LS8 cells during one-day cycle of 24 h by next generation deep sequencing. After removal of low quality reads, contaminants, and ligation products, we obtained a high number of clean reads in all 12 samples from four different time points. The length distribution analysis indicated that 77.5% of clean reads were between 21 and 24 nucleotides (nt), of which 35.81% reads exhibited a length of 22 nt. In total, we identified 1471 miRNAs in LS8 cells throughout all four time-points. 1330 (90.41%) miRNAs were identified as known miRNA sequences, whereas 139 (9.59%) were unannotated and classified as novel miRNA sequences. The differential expression analysis showed that 191 known miRNAs exhibited significantly (P-value < 0.01) different levels of expression across three time-points investigated (T6, T12, and T18) compared to T0. Verification of sequencing data using qRT-PCR on six selected miRNAs suggested good correlation between the two methods. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed significant enrichment of predicted target genes of differentially expressed miRNAs. The present study shows that miRNAs are highly expressed in LS8 cells and that a significant number of them oscillate during one-day cycle of 24 h. This is the first transcriptomic analysis of miRNAs in ameloblast-like cell line LS8 that can be potentially used to further characterize the epigenetic regulation of miRNAs during enamel formation.