Deciphering the Role of Putative Novel miRNAs Encoded From the Newly Found Genomic Regions of T2T-CHM13 in the Progression of Collecting Duct Renal Cell Carcinoma

Abstract

Background: Collecting duct renal cell carcinoma (cdRCC) is a rare and aggressive renal cancer subtype. The molecular mechanisms underlying cdRCC remain poorly understood, which presents a significant challenge for the development of effective treatment strategies. Advances in genome sequencing, particularly the discovery of new genomic regions in the T2T-CHM13 reference genome, have provided an opportunity to expand our understanding of this disease.

Aims: Our study specifically aims to investigate the role of miRNAs encoded by these regions, proposing them as potential epigenetic regulators in the pathogenesis of cdRCC.

Methods: We used bioinformatic pipelines and small RNA-seq data analysis to predict novel miRNAs from the newly discovered genomic regions of T2T-CHM13. RNA-seq analysis of cdRCC tumors was performed to identify differentially expressed genes, and target prediction tools were used to find miRNA-mRNA interactions. Functional enrichment analyses were conducted to characterize the biological pathways affected.

Results and discussion: Using computational approaches, we predicted 156 novel miRNAs from T2T-CHM13's newly resolved sequences. RNA-seq and miRNA-mRNA interaction analyses identified 345 downregulated genes targeted by novel miRNAs and 395 downregulated genes targeted by known miRNAs. A comprehensive functional enrichment analysis of these perturbed genes revealed distinctive pathways, including cGMP-PKG signaling, calcium signaling, adipocytokine signaling, PPAR signaling, and apelin signaling, all of which are implicated in tumorigenesis. Furthermore, Gene Ontology analysis linked miRNA-targeted genes to disrupted cell-cell junctions and adhesion, providing a mechanistic explanation for aggressive invasion and metastasis in cdRCC. Additionally, a significant number of the target genes involved in metabolic and ion transport pathways were perturbed, explaining metabolic alterations in the cancer cells. We also identified 15 tumor suppressor genes downregulated by novel miRNAs, 6 of which were uniquely targeted, highlighting the potential of these miRNAs as cdRCC-specific biomarkers.

Conclusion: In conclusion, our study offers valuable insights into cdRCC biology from an epigenetic perspective, laying the groundwork for future research aimed at developing targeted therapies.

Keywords: T2T‐CHM13; cancer; carcinoma; collecting duct; epigenetic modulator; miRNA; novel; tumor‐suppressor.

FIGURE 1

Workflow of identification of novel putative human miRNAs and understanding their role in cdRCC by analyzing their target genes.

FIGURE 2

RNA‐seq analysis of collecting duct renal cell carcinoma (cdRCC) versus normal samples. (A) Principal component analysis (PCA) plot of cdRCC versus normal samples, showing a clear separation between the two groups. (B) Volcano plot of differentially expressed genes between cdRCC and normal samples, with differentially expressed genes in red (adjusted p‐value < 0.05 and |log2FoldChange| > 2) and non‐differentially expressed genes in blue (adjusted p‐value < 0.05 and |log2FoldChange| ≤ 2) and black (adjusted p‐value ≥ 0.05 and |log2FoldChange| ≤ 2). Genes represented in red with a positive log2FoldChange are upregulated, while those with a negative log2FoldChange are downregulated. (C) Heatmap of the differentially expressed genes between cdRCC and normal samples showing the relative expression levels of these genes across all samples, with red indicating high expression and blue indicating low expression. The abbreviation “Tu” represents tumor samples, while “No” represents normal samples.

FIGURE 3

Network analysis of miRNA‐target gene interactions and identification of downregulated target genes in collecting duct renal cell carcinoma (cdRCC). (A) Network diagram of the interactions between novel miRNAs and their target genes. (B) Network diagram of the interactions between novel miRNAs and their target genes that are downregulated in cdRCC. (C) Network diagram of the interactions between previously known miRNAs and their target genes that are downregulated in cdRCC. (D) Venn diagram of the overlap between the novel miRNA target genes and previously known miRNA target genes that are downregulated in cdRCC.

FIGURE 4

Results of enrichment analyses of miRNA target genes that are downregulated in cdRCC. Each heatmap in the figure represents the results of enrichment analyses for miRNA target downregulated genes in cdRCC including selected, (A) KEGG pathways, (B) GOMF terms, (C) GOCC terms, and (D) GOBP terms. Each heatmap is divided into three columns, namely “Novel”, representing novel miRNA target genes, “Known”, representing previously known miRNA target genes, and “Both”, representing both miRNA target genes. The color intensity in the heatmaps indicates the significance level, with colors tending towards red indicating more significance, yellow indicating less significance, and gray indicating non‐significant enrichment.

FIGURE 5

A heatmap of selected downregulated tumor suppressor genes in cdRCC, indicating whether they are targeted by novel or previously known miRNAs. Blue cells indicate targeting by the corresponding miRNA set, while gray cells indicate non‐targeting.