Distinct microRNAs expression profile in primary biliary cirrhosis and evaluation of miR 505-3p and miR197-3p as novel biomarkers

Abstract

Background and aims: MicroRNAs are small endogenous RNA molecules with specific expression patterns that can serve as biomarkers for numerous diseases. However, little is known about the expression profile of serum miRNAs in PBC.

Methods: First, we employed Illumina deep sequencing for the initial screening to indicate the read numbers of miRNA expression in 10 PBC, 5 CH-C, 5 CH-B patients and 5 healthy controls. Comparing the differentially expressed miRNAs in the 4 groups, analysis of variance was performed on the number of sequence reads to evaluate the statistical significance. Hierarchical clustering was performed using an R platform and we have found candidates for specific miRNAs in the PBC patients. Second, a quantitative reverse transcription PCR validation study was conducted in 10 samples in each group. The expression levels of the selected miRNAs were presented as fold-changes (2(-ΔΔCt)). Finally, computer analysis was conducted to predict target genes and biological functions with MiRror 2.0 and DAVID v6.7.

Results: We obtained about 12 million 32-mer short RNA reads on average per sample and the mapping rates to miRBase were 16.60% and 81.66% to hg19. In the statistical significance testing, the expression levels of 81 miRNAs were found to be differentially expressed in the 4 groups. The heat map and hierarchical clustering demonstrated that the miRNA profiles from PBC clustered with those of CH-B, CH-C and healthy controls. Additionally, the circulating levels of hsa-miR-505-3p, 197-3p, and 500a-3p were significantly decreased in PBC compared with healthy controls and the expression levels of hsa-miR-505-3p, 139-5p and 197-3p were significantly reduced compared with the viral hepatitis group.

Conclusions: Our results indicate that sera from patients with PBC have a unique miRNA expression profile and that the down-regulated expression of hsa-miR-505-3p and miR-197-3p can serve as clinical biomarkers of PBC.

Figure 1. Heat map and hierarchical clustering.

Individual miRNA expression were calculated by R platform and heat map was computed and described using a function of heatmap.2 in gplots. It uses hierarchical clustering with Euclidean distance; Pearson Linear Correlation and Ward's method to generate the hierarchical tree . ANOVA was applied to extract differentially expressed miRNAs and adjustment of the p-value by multiple comparisons was performed by calculating FDR. Those miRNAs with FDR<0.1 were presented. The red indicates high level of miRNA expression and the blue shows low.

Figure 2. Validation of deep sequencing results for selected miRNAs.

We have registered 10 samples in each group listed on Table S3. The threshold cycle for each miRNA primer/probe set were normalized with spiked in cel-miR-39 primer/probe pair and compared to CH-C-5. Result for normally distributed continuous variables are given as means and compared between groups by Student's t-test. Results for non-normally distributed continuous variables are summarized as medians and were compared by Mann-Whitney U test. Statistical significance indicates by one asterisk (p<0.05) and two (p<0.01).