Differentially expressed miR-3680-5p is associated with parathyroid hormone regulation in peritoneal dialysis patients

<div><p>Mineral and bone disorder (MBD) is observed universally in patients with chronic kidney disease (CKD). Detrimental MBD-related skeletal changes include increased prevalence of fracture, cardiovascular disease, and mortality. MicroRNAs (miRNAs) have been identified as useful biomarkers in various diseases, and the aim of this study was to identify miRNAs associated with parathyroid hormone level in peritoneal dialysis (PD) patients. Fifty-two PD patients were enrolled and grouped by their intact parathyroid hormone (iPTH) level; 11 patients had low iPTH (<150 pg/mL) and 41 patients had high iPTH (≥150 pg/mL). Total RNA was extracted from whole blood samples. Total RNA from 15 patients (7 and 8 patients in the low and high iPTH groups, respectively) underwent miRNA microarray analysis, and three differentially upregulated (>2-fold change) miRNAs previously associated with human disease were selected for real-time quantitative PCR (qPCR) analysis. Interaction analyses between miRNAs and genes were performed by using TargetScan and the KEGG pathway database. Microarray results revealed 165 miRNAs were differentially expressed between patients with high iPTH levels and low iPTH levels. Of those miRNAs, 81 were upregulated and 84 were downregulated in patients with high iPTH levels. Expression levels of miR-1299, miR-3680-5p, and miR-548b-5p (previously associated with human disease) in 52 patients were analyzed by using qPCR. MiR-3680-5p was differentially expressed in low and high iPTH patients (<i>P</i> < 0.05). The predicted target genes of miR-3680-5p were <i>USP6</i>, <i>USP32</i>, <i>USP46</i>, and <i>DLT</i>, which are involved in the ubiquitin proteolysis pathway. This pathway has roles in PTH and parathyroid hormone related protein degradation and proteolysis. The mechanisms involved in the associations among low PTH, adynamic bone disease, miR-3680-5p, and the target genes should be explored further in order to elucidate their roles in CKD-MBD development.</p></div

Volcano plot for differentially expressed miRNAs in patients with low (<150 pg/mL) and high (≥150 pg/mL) iPTH levels.

<p>Volcano plot for differentially expressed miRNAs in patients with low (<150 pg/mL) and high (≥150 pg/mL) iPTH levels.</p

Heat map illustrating miRNAs profiles in patients with low (<150 pg/mL) and high (≥150 pg/mL) iPTH levels.

<p>The log₂ values were calculated for each sample by normalizing to the count number of reads alone. The heat map analysis was performed by using Cluster 3.0 with the Euclidean distance algorithm and average linkage (<i>P</i>_adj <0.05 and log₂ fold change >2). Group 1: iPTH < 150 pg/mL, Group 2: iPTH ≥ 150 pg/mL</p

Demographic and baseline biochemical parameter characteristics of study participants.

<p>Demographic and baseline biochemical parameter characteristics of study participants.</p

Pathways and genes predicted to be associated with miR-3680-5p.

<p>Pathways and genes predicted to be associated with miR-3680-5p.</p

Histogram of sample sizes from 654 drug-related SNPs.

<p><b>A</b>. Total sample sizes of SNPs. <b>B</b>. Sample size of each population of SNPs. CHB and JPT are plotted separately according to the format of the original HapMap Data. SNPs with larger sample sizes are included in Phase III, and SNPs with smaller sample sizes are included in Phase II.</p

Q-values and fold enrichments of significant terms in HD group.

<p>FE: Fold enrichment</p><p>BH’s q: Benjamini-Hochberg’s q-value</p><p>Q-values and fold enrichments of significant terms in HD group.</p

Sensitivities (%) of each measure from simulation data under H₀:<i>d</i>=0.05,0.1…,0.3 due to bias in sample size (Scenario II).

<p>A. (<i>n</i>₁,<i>n</i>₂,<i>n</i>₃) = (200,100,100). <b>B</b>. (<i>n</i>₁,<i>n</i>₂,<i>n</i>₃) = (100,200,100). <b>C</b>. (<i>n</i>₁,<i>n</i>₂,<i>n</i>₃) = (100,100,200). Blue line: chi-square test; red line: F_st; black dotted line: ANOVA F-test; green dotted line: SS_d from NSCM.</p