Supplementary Material - Study: Human transcriptome DEGs - decayed vs healthy teeth

Description

The rapid progression of decayedcarious lesions in dentin is facilitated by its reduced mineralization, high organic content, and tubular structure, which contribute to extracellular matrix degradation through MMP activation in acidic conditions. This study uses RNA-Seq to explore these processes in decayed teeth, focusing on the differential expression of genes potentially involved in extracellular matrix degradation, including analysis in the pulp, predentin, and dentin, and the expression of collagenases (MMP-2 and MMP-9) and their preferred inhibitors (TIMP-1 and TIMP-2). RNA was sequenced using Illumina® technology, followed by quality validation, read alignment, and differential gene expression analysis. Gene Ontology and KEGG pathway analyses identified significant biological processes. Sequencing produced 16-37 million reads with 87.25%-88.08% alignment, identifying 1% of over 33,000 genes as differentially expressed. Upregulated genes in decayed tissues are linked to detoxification and stress responses, while downregulated genes relate to energy metabolism and tooth development. Interaction network analysis highlights proteins like EGFR and metallothioneins in acute-phase responses. MMP-2 is highly expressed in decayed predentin and dentin, while MMP-9 is reduced. TIMP-1, mainly in decayed dentin, aligns with its role as an MMP-9 inhibitor. TIMP-2, which inhibits MMP-2, shows elevated levels in both decayed and caries-free dentin. Upregulation of inflammatory and stress-related genes underscores their role in maintaining extracellular matrix integrity. keywords: Cariology; Gene Expression; Matrix metalloproteinases; Dentin; Inflammation; Oxidative stress; RNA-Seq.

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Deep sequencing was performed with the Illumina® NextSeq 1000/2000 P2 kit (200 cycles), generating approximately 40 million reads per sample and providing sufficient data for target enzymes’ analysis. The High Throughput Sequencing Platform at Instituto Oswaldo Cruz/Fiocruz, Brazil, was responsible for library preparation, sequencing, adapter removal, multiplexing provision of FASTQ files. Sequencing data quality was initially evaluated with FASTQC, followed by low-quality bases/sequences removal with TRIMMOMATIC (Bolger et al., 2014). The set parameters included a minimum PHRED quality score of 28 and a minimum length of 70 base pairs per read. Sequenced data were then aligned to the human transcriptome (CHR38 version) using SALMON (Patro et al., 2017) for quantification and normalization utilizing the Transcripts Per Million (TPM) index. Gene expression and differential transcript analysis were conducted using R software (R Core Team, 2021), incorporating DESeq2 package (Love et al., 2014), adhering to a FDR < 0.05 and |log2FC| >= 1 criteria.

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