MicroRNAs as a therapeutic target in IgA nephropathy in Indian population

Abstract

Immunoglobulin A nephropathy (IgAN) is the most frequent glomerular disease with rapid development to end stage renal disease, requiring renal replacement therapy. Genome-wide studies suggest geographical variations in genetic susceptibility to IgAN and disease progression. Specific 'candidate genes' were indicated to correlate with different functions that are involved in the pathogenesis of renal conditions. MicroRNAs (miRNAs/miRs) have a major role in mRNA degradation or translation repression, thereby regulating the expression of their target proteins. Previously, a small number of miRNAs were reported to have direct associations with IgAN. In the present study, new miRNAs linked to IgAN were identified in the Indian population. The miRNA was isolated from kidney biopsies of patients with IgAN (n=6) and healthy control tissue from patients with renal cell carcinoma (n=6). The sequencing results indicated that the miRNA percentage acquired from controls and patients with IgAN was 5.61 and 4.35%, respectively. From the results, 10 upregulated and 15 downregulated miRNAs were identified. Of the 25 differentially expressed miRNAs (DEMs), miR-181a-5p, miR-28-3p, let-7g-5p, miR-92a-3p and miR-30c-5p were not reported previously. Furthermore, Kyoto Encyclopedia of Genes and Genomes and Gene Ontology analyses suggested that the target genes of the DEMs were mainly enriched in pathways such as cancer, ErbB signalling, proteoglycans in cancer, Hippo signalling and MAPK pathways. The newly identified miRNAs may impact the behaviour of tissues or IgA deposition by regulating signalling pathways, which forms a basis for future studies aimed at improving the diagnosis and care of patients with IgAN in the Indian community.

Keywords: immunoglobulin A nephropathy; kidney biopsy; microRNA; next generation sequencing.

Figure 1

miRNA sequencing analysis in healthy participants and IgAN patients. (A) The workflow of the miRNA sequencing experiment. (B and C) Read length distributions of miRNA in (B) healthy participants and (C) patients with IgAN. (D-H) Alignment percentage in each sample along with (D) alignment breakdown, (E) total read count, (F) coverage read counts, (G) average base quality score per position and (H) per reads. (I and J) Quantification of coverage breakdown in both healthy participants (C1, C2….C6) and patients with IgAN (P1, P2…P6). miRNA, microRNA; IgAN, immunoglobulin A nephropathy; HC, healthy controls.

Figure 2

Differentially expressed miRNA profiles in healthy participants and patients with IgAN. (A) Scatter plot of three-dimensional PCs of the data for patients with IgAN and healthy controls. (B) Heat map of significantly differentially expressed miRNAs in healthy controls as well as in patients with IgAN. The red color indicates high expression level and blue color indicates lower expression levels (FC ≥2 or ≤0.5 and P≤0.05). (C) Volcano plot indicating numbers of upregulated and downregulated miRNAs in patients with IgAN as compared to healthy controls. Each point in the plot represents a gene and Log2-fold change and statistical significance (P≤0.05) of each gene was calculated based on differential gene expression analysis. Red points indicate significantly upregulated genes and blue points indicate significantly downregulated genes. The x-axis indicates the fold change with no significant change as the mid-point. (D) Pie chart of distributions of significantly differentially expressed upregulated and downregulated miRNAs in patients with IgAN (P1, P2…P6) compared with healthy controls (C1, C2…C6). miRNA, microRNA; IgAN, immunoglobulin A nephropathy; PCA, principal component analysis; N/C, no significance.

Figure 3

Regulatory network of upregulated miRNAs and their target genes. Red circles indicate the miRNAs, blue circles represent target genes and the lines indicate the interaction between miRNA and their target genes. miRNA/miR, microRNA.

Figure 4

Illustration of Gene Ontology enrichment and KEGG pathway analysis for the top 10 upregulated miRNAs. Gene Ontology enrichment analysis in the categories of (A) Biological Process, (B) Cellular Component and (C) Molecular Function. (D) Top 20 enriched KEGG pathways of potential target genes for the upregulated miRNAs. The size of the points in the graph indicates the number of differential genes enriched and the color indicates the FDR value. FDR, false discovery rate; KEGG, Kyoto Encyclopedia of Genes and Genomes; miRNA, microRNA.

Figure 5

Regulatory network of downregulated miRNAs and their target genes. Yellow circles represent the miRNAs, blue circles represent the downregulated target genes and the lines indicate the interaction between miRNAs and their target genes. miRNA/miR, microRNA.

Figure 6

Illustration of Gene Ontology enrichment and KEGG pathway analysis for the top 15 downregulated miRNAs. Gene Ontology terms in the categories of (A) Biological Process, (B) Cellular Component and (C) Molecular Function. (D) Top 20 enriched KEGG pathways of potential target genes for the downregulated miRNAs. The size of the points in the graph indicates the number of differential genes enriched and the color indicates the FDR value. miRNA, microRNA; FDR, false discovery rate; KEGG, Kyoto Encyclopedia of Genes and Genomes.