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DISTINCT SALIVARY PROTEOMIC PATTERNS AS SIGNATURE FOR MOLAR INCISOR HYPOMINERALIZATION STAGES

ABSTRACT: Saliva is a rich-bodily fluid with recognized clinical diagnosis roles and this research aimed at investigating if there is any change in the salivary proteome signatures of MIH children with distinct degrees of severity. Fifty schoolers (6-10 years) were equally assigned into the following groups: Group 1 (Control group - Healthy teeth), Group 2 (Mild MIH with white/creamy opacity and free of caries), Group 3 (Mild MIH with yellow/brown opacity and free of caries), Group 4 (Severe MIH with white/creamy, yellow/brown opacities including post-eruptive fracture and free of caries), Group 5 (Severe MIH with white/creamy, yellow/brown opacities, post-eruptive fracture, and caries). Unstimulated saliva samples were collected and later explored using mass spectrometry analysis. In total, 6,471 proteins were found, 5,073 exclusively from MIH children, and 778 overlapping among the different degrees of the disturb. The Biological Pathways displayed distinct patterns among the groups, being different according to the degrees of MIH. Gene-Odontology differences might not be verified regarding the biological processes and cellular components. Conversely, with respect to molecular function, alterations among groups were evident, with the presence of proteins that would contribute to MIH in children with the severe condition (i.e, calcium ion binding, microtubule binding, platelet-derived growth factor binding). In conclusion, the results of this study support important salivary proteomic changes in MIH children, according to distinct degrees of severity, reinforcing the interplay between the clinical aspects of hypomineralization (white/yellow opacities/enamel breakdown) and changes in the salivary proteome.

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Thais Manzano Parisotto

PROVIDER: PXD044610 | JPOST Repository | Sun Aug 18 00:00:00 BST 2024

REPOSITORIES: jPOST

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Project description:Saliva (oral fluids) is an emerging biofluid poised for clinical diseases detection. Although the rationale for oral diseases applications (e.g. oral cancer) is clear, the rationale and relationship between systemic diseases and saliva biomarkers are unknown. In this study, we used mouse models of melanoma and non-small cell lung cancer and compared the transcriptome biomarker profiles of tumor-bearing mice to those of control mice. Microarray analysis showed that salivary transcriptomes were significantly altered in tumor-bearing mice vs. controls. Analysis of the transcriptomes in the mouse tumors, serum, salivary glands and saliva revealed that salivary biomarkers have multiple origins. Furthermore, we identified that the expression of two groups of significantly altered transcription factors Runx1, Mlxipl, Trim30 and Egr1, Tbx1, Nr1d1 in melanoma-bearing mice that can potentially be responsible for 82.6% of the up-regulated genes expression and 62.5% of the down-regulated gene expression in the mice saliva, respectively. We also confirmed that the ectopic production of nerve growth factor (NGF) in the melanoma tumor tissue as a tumor-released mediator that can induce expression of the transcription factor Egr-1 in the salivary gland. Taken together, our data support the conclusion that upon systemic disease development, a disease-specific change occurs in the salivary biomarker profile. Although the origins of the disease-specific salivary biomarkers are both systemic and local, stimulation of salivary gland by mediators released from remote tumors play an important role in regulating the salivary surrogate biomarker profiles. Experiment Overall Design: Mice (either C57BL/6 mice or DBA/2 mice) were randomly assigned to control group and tumor group (15 animals per group). Melanoma mice model was induced by subcutaneous (s.c.) injection of B16-F1 cells in 0.1 ml PBS into the lower-right flank of C57BL/6 mice. The lung cancer model was established by s.c. injection of KLN-205 cells in DBA/2 mice. Control animal were injected with PBS alone. Established tumors were observed after 2-3 weeks. Experiment Overall Design: When tumors reached 15 mm in diameter saliva was collected and the mice were sacrificed. Mild anesthesia was induced. Mice saliva was stimulated, obtained and immediately placed in pre-schilled 1.5-ml microcentrifuge tubes. Collection was completed in 20 minutes and samples were stored at -80ÂºC until analyzed.In addition, Blood was collected in BD Vacutainer tubes containing clot activator (BD Biosciences). Salivary gland and tumor tissue were removed from mice, snap-frozen in liquid nitrogen and stored at -80ÂºC. Experiment Overall Design: Experiment Overall Design: Salivary,salivary gland, serum or tumor RNA was isolated using the RNeasy Mini Kit (Qiagen) as described previously. There are 15 mice in the control group or tumor group (totally 30 C57BL/6 mice for melanoma mouse model, another 30 DBA/2 mice for lung cancer mouse model). Samples derived from 5 mice in each group were pooled and RNA extracted. The pooling is necessary to ensure sufficient salivary mRNA can be obtained for microarray analyses. Isolated total RNA was treated with recombinant DNase (Ambion, Austin, TX). For microarray analysis, mRNA from mouse saliva, gland or tumor was linearly amplified using the RiboAmp RNA Amplification kit (Molecular Devices, Sunnyvale, CA). After purification, cDNA were in vitro transcribed and biotinylated using GeneChip Expression 3â-Amplification Reagents for in vitro transcription labeling (Affymetrix, Santa Clara, CA). The labeled RNAs was subsequently fragmented, hybridization and scanning.

Project description:Background: We seek to understand correlates of chronic stress and their influence on the development of substance use in a population-based cohort of young adults (OYSUP). Methodology: We evaluated clinical features, salivary RNA metrics and differential expression of candidate genes in unfractionated saliva samples from 48 individuals (31% female, 55% ever-smoking) randomly selected from two groups stratified by high versus low severe negative life events and difficulties within the last year as as measured by the Life Events and Difficulties Schedule (LEDS, Brown & Harris, 1978). We used multiple analytical platforms, multiple reference genes and 18 replicates of all gene expression assays (assays). We chose 38 candidate genes previously identified as differentially expressed in a genome-wide scan of monocyte RNA in 11 familial caregivers of glioblastoma multiforme patients and 10 control subjects matched on age (middle-aged), gender (57% female), ethnicity and marital status and free of major stressors during the previous year. Principal Findings: We observed significant differences in smoking prevalence (Pearson’s ?22d.f.=4.5, P=0.035) and RNA integrity score (t=2.12, P=0.039) between stress strata. We analyzed quantitative PCR data using both the comparative standard curve (CSC) and relative standard curve (RSC) methods because 34% of assays had efficiency estimates <90%. Assay data exhibited excellent linearity and low variability. We observed significant under expression of five assays in the high stress stratum using both methods, four of which interrogate glucocorticoid receptor regulated genes. Post-hoc analyses identified significant associations of RNA yield, integrity score, gender, ever-smoking and/or stress with results of normalized gene expression in 22/37 assays. IL8 expression remained significantly associated with chronic stress after adjustment for clinical and salivary RNA variables. Salivary IL8 expression has been previously associated with oropharyngeal squamous cell cancer. Conclusions: A gene expression signature of chronic stress previously observed in adult hematopoietic samples is observed in the unfractionated saliva of young adults. Future investigation will need to analyze associated clinical, metabolic, meta-genomic, and proteomic components of unfractionated saliva to elucidate factors influencing the salivary transcriptome. Gene expression was performed on a Fluidigm 48 x 48 array on 48 individuals for 38 target genes and 6 possible reference genes (represented by 9 reference assays) in triplicate. Quantitative Q-PCR datas were normalized according to relative levels of four reference genes (CDKN1B, YWHAZ, RPL13A, and UBC).

Project description:Background: We seek to understand correlates of chronic stress and their influence on the development of substance use in a population-based cohort of young adults (OYSUP). Methodology: We evaluated clinical features, salivary RNA metrics and differential expression of candidate genes in unfractionated saliva samples from 48 individuals (31% female, 55% ever-smoking) randomly selected from two groups stratified by high versus low severe negative life events and difficulties within the last year as as measured by the Life Events and Difficulties Schedule (LEDS, Brown & Harris, 1978). We used multiple analytical platforms, multiple reference genes and 18 replicates of all gene expression assays (assays). We chose 38 candidate genes previously identified as differentially expressed in a genome-wide scan of monocyte RNA in 11 familial caregivers of glioblastoma multiforme patients and 10 control subjects matched on age (middle-aged), gender (57% female), ethnicity and marital status and free of major stressors during the previous year. Principal Findings: We observed significant differences in smoking prevalence (Pearson’s χ22d.f.=4.5, P=0.035) and RNA integrity score (t=2.12, P=0.039) between stress strata. We analyzed quantitative PCR data using both the comparative standard curve (CSC) and relative standard curve (RSC) methods because 34% of assays had efficiency estimates <90%. Assay data exhibited excellent linearity and low variability. We observed significant under expression of five assays in the high stress stratum using both methods, four of which interrogate glucocorticoid receptor regulated genes. Post-hoc analyses identified significant associations of RNA yield, integrity score, gender, ever-smoking and/or stress with results of normalized gene expression in 22/37 assays. IL8 expression remained significantly associated with chronic stress after adjustment for clinical and salivary RNA variables. Salivary IL8 expression has been previously associated with oropharyngeal squamous cell cancer. Conclusions: A gene expression signature of chronic stress previously observed in adult hematopoietic samples is observed in the unfractionated saliva of young adults. Future investigation will need to analyze associated clinical, metabolic, meta-genomic, and proteomic components of unfractionated saliva to elucidate factors influencing the salivary transcriptome.

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DISTINCT SALIVARY PROTEOMIC PATTERNS AS SIGNATURE FOR MOLAR INCISOR HYPOMINERALIZATION STAGES

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