Comprehensive Profiling of the Circulatory miRNAome Response to a High Protein Diet in Elderly Men: A Potential Role in Inflammatory Response Modulation.
Author(s): Ramzan F, Mitchell CJ, Milan AM, Schierding W, Zeng N, Sharma P, Mitchell SM, D'Souza RF, Knowles SO, Roy NC, Sjödin A, Wagner KH, Cameron-Smith D
Publication: Mol Nutr Food Res, 2019, Vol. 63, Page e1800811
PubMed ID: 30892810 PubMed Review Paper? No
Purpose of Paper
The purpose of this paper was to investigate the effects of a high protein diet on the circulating microRNA (miRNA, miR) profile and the expression of a subset of RNA in older men (≥70 y).
Conclusion of Paper
Of the 179 miRNAs identified after filtering, only miR-125b-5p, miR-100-5p, miR-99a-5p, miR-23b-3p, and miR-203a were down-regulated (>2-fold change, P adjusted ≤0.05) in plasma of patients on the two times recommended dietary allowance (2RDA) of protein diet compared to the recommended dietary allowance (RDA) of protein diet. Of these five miRNAs, real-time PCR only confirmed downregulation of miR-125b-5p and miR-23b-3p after consumption of the 2RDA diet and real-time PCR levels were not correlated with sequencing levels for three of five miRNAs. Analysis of potential RNA targets for these five miRNAs in white cells showed significantly higher expression of the inflammatory markers phosphatase and tensin homolog (PTEN), interleukin 6 (IL-6), tumor necrosis factor (TNF-α), and interleukin 8 (IL-8) and lower expression of homeobox A1 (HOXA1) and protein phosphatase 1 catalytic subunit beta (PPP1CB) in the 2RDA group than the RDA group.
Studies
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Study Purpose
The purpose of this study was to investigate the effects of a high protein diet on the circulating miRNA profile and the expression of select miRNA targets in older men (≥70 y). A total of 29 healthy, male non-smokers over 70 years of age were randomly assigned to either consume delivered meals containing 0.8 g protein/kg a day (RDA, n=15) or 1.6 g protein/kg a day (2 times RDA n=14) for 10 weeks. Participants remained blind to their assigned group. Fasting blood was collected into EDTA and heparin Vacutainer tubes pre-diet change and after 10 weeks of participation. EDTA plasma was obtained by centrifugation at 1900 × g for 15 min at 4°C within 2 h of storage at 4°C and subsequently stored at -80°C. Hemolysis was evaluated visually. RNA was extracted using the Plasma/Serum RNA Purification Mini Kit. Sequencing libraries were constructed using the Small RNA Library Prep Kit for Illumina, sequenced on an Illumina HiSeq 4000, and analyzed using the miA RNA-seq pipeline in a Bioconductor using thresholds of ≥15 counts per million in 75% of samples analyzed and pairwise comparisons were performed using DEseq2. RNA was reverse-transcribed using TaqMan Advanced miRNA cDNA Synthesis Kit and quantified using custom TaqMan MicroRNA assays. White cells were isolated from heparinized blood using the Ficoll-Paque method and RNA was isolated using the AllPrep DNA/RNA/miRNA Universal Kit. RNA was reverse-transcribed using the High Capacity RNA-to-cDNA Kit and levels of HOXA1, DEAH-box helicase 33 (DHX33), RB transcriptional corepressor 1 (RB1), ribosomal protein L7 like 1 (RPL7L1), PPP1CB, insulin like growth factor 1 receptor (IGF1R), AKT Serine/Threonine kinase 1 (AKT1), trinucleotide repeat containing 6B (TNRC6B), vascular endothelial growth factor (VEGF), forkhead box O1 (FOXO1), PTEN, IL-6, TNF-α, and IL-8 were quantified by real-time PCR.and IL-8 were quantified by real-time PCR.
Summary of Findings:
While 1173 different miRNAs were represented across the samples, the 50 most abundant miRNAs represented 97.9% of the total miRNA reads. Of the 179 miRNAs identified after filtering (≥15 counts per million in 75% of samples analyzed), five (miR-125b-5p, miR-100-5p, miR-99a-5p, miR-23b-3p, and miR-203a) were down-regulated (>2-fold change, P adjusted ≤0.05) in plasma of patients on the 2RDA diet compared to the RDA diet. Of these five miRNAs, real-time PCR only confirmed downregulation of miR-125b-5p and miR-23b-3p after consumption of the 2 RDA diet. Further, the sequenced miRNA reads were significantly correlated with PCR abundances for only two of the five initially identified miRNAs: miR-99a-5p (r=0.32, P=0.04) and miR-125b-5p (r=0.48, P<0.01). The five miRNAs identified by sequencing were predicted to regulate 1298 genes, 12 of which were strongly regulated by three or more of the miRNAs. These 12 genes are involved in lipid metabolic process and negative regulation of signal transduction, phosphorylation, phosphate metabolic process, and response to stimulus. Additional potential genes were identified using these pathways. Analysis of white cells showed the 2RDA group to have higher mRNA levels of pTEN (P=0.016), IL-6 (P<0.001), TNF-α (P=0.030), and IL-8 (P<0.001) and lower levels of HOXA1 (P=0.022) and PPP1CB (P=0.008) than the RDA group and no difference in DHX33, RB1, VEGF, IGF1R, or AKT1 levels.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Normal
Platform:
Analyte Technology Platform RNA Real-time qRT-PCR RNA Next generation sequencing Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Preaquisition Patient diet 0.8g protein/kg a day
1.6 g protein/kg a day
Real-time qRT-PCR Specific Targeted nucleic acid HOXA1
PPP1CB
pTEN
IL-6
IL-8
TNF-α
AKT1
IGF1R
VEGF
miR-125b-5p
miR-100-5p
miR-99a-5p
miR-23b-3p
miR-203a
DHX33
RB1
Real-time qRT-PCR Specific Technology platform NGS