The Integrative Studies on the Functional A-to-I RNA Editing Events in Human Cancers

Abstract

Adenosine-to-inosine (A-to-I) RNA editing, constituting nearly 90% of all RNA editing events in humans, has been reported to contribute to the tumorigenesis in diverse cancers. However, the comprehensive map for functional A-to-I RNA editing events in cancers is still insufficient. To fill this gap, we systematically and intensively analyzed multiple tumorigenic mechanisms of A-to-I RNA editing events in samples across 33 cancer types from The Cancer Genome Atlas. For individual candidate among ∼ 1,500,000 quantified RNA editing events, we performed diverse types of downstream functional annotations. Finally, we identified 24,236 potentially functional A-to-I RNA editing events, including the cases in APOL1, IGFBP3, GRIA2, BLCAP, and miR-589-3p. These events might play crucial roles in the scenarios of tumorigenesis, due to their tumor-related editing frequencies or probable effects on altered expression profiles, protein functions, splicing patterns, and microRNA regulations of tumor genes. Our functional A-to-I RNA editing events (https://ccsm.uth.edu/CAeditome/) will help better understand the cancer pathology from the A-to-I RNA editing aspect.

Keywords: A-to-I RNA editing; Alternative splicing; Cancer; MicroRNA regulation; Protein recoding.

Figure 1

The flowchart to identify functional A-to-I RNA editing events in cancers A. The collection and pre-processing of multi-omics data across 33 cancer types. B. Diverse down-stream analyses of A-to-I RNA editing events. C. The potentially functional A-to-I RNA editing events related to tumorigenesis. A-to-I, adenosine-to-inosine; TCGA, The Cancer Genome Atlas; RNA-seq, RNA sequencing; SNP, single-nucleotide polymorphism; miRNA, microRNA; 3′-UTR, 3′-untranslated region; lncRNA, long non-coding RNA; TSG, tumor suppressor gene; GTEx, Genotype-Tissue Expression; WT, wild-type.

Figure 2

RNA editing frequency analysis A. KIRC-specific A-to-I RNA editing events with more than five edited tumor samples and non-edited normal controls. The histogram presents the distribution of this kind of RNA editing events along with the number of edited tumor samples. B. The bubble plot introduces a part of KIRC-specific RNA editing events in TGs which are not edited in normal controls. C. One significant case of IGFBP3 occurred only in 246 tumor samples for the KIRC cancer type, also showing higher editing frequencies in more severe KIRC tumors. D. KIRC-specific A-to-I RNA editing events showing differential editing frequencies in KIRC tumors compared with controls. The volcano plot presents the differences of editing frequencies between tumor samples and controls. E. The bubble plot introduces a part of KIRC-specific RNA editing events in TGs which are differentially edited in tumor samples compared with normal controls. F. One significant case in APOL1 showed higher editing frequencies in KIRC tumor samples. G. KIRC stage-associated A-to-I RNA editing events. The volcano plot presents the correlations of editing frequencies with tumor stages. H. The bubble plot introduces a part of KIRC stage-associated RNA editing events in TGs. I. One significant case in APOL1 showed higher editing frequencies in more severe KIRC tumors. J. KIRC survival-related A-to-I RNA editing events. The volcano plot presents the correlations of editing frequencies with cancer survival. K. The bubble plot introduces a part of KIRC survival-related RNA editing events in TGs. L. One significant case in APOL1 showed higher editing frequencies in the poorer survival group. The analysis results of editing frequencies for other cancer types are displayed in Figures S1–S8. KIRC, kidney renal clear cell carcinoma; TG, tumor gene; HR, hazard ratio.

Figure 3

The effects of A-to-I RNA editing events on gene expression A.APOL1 was up-regulated in the RNA-edited group. B. The expression levels of APOL1 were positively associated with the frequencies of the CAediting_1478179 editing event. C.APOL1 was abnormally expressed in KIRC tumor samples compared with controls. D. The CAediting_1478179 editing event seems to be a potential biomarker for the KIRC cancer type, because it caused the loss of original miRNA binding targets to induce the up-regulated expression of APOL1, which may interfere in the autophagy function of this gene in cancer. E. The analysis procedure for the effects of A-to-I RNA editing events on gene expression. First, we performed a DEG analysis between RNA-edited and non-edited tumor samples, as well as a correlation analysis between gene expression and editing frequency to identify the DEGs whose expression levels were probably affected by A-to-I RNA editing. Then, we overlapped these genes with the DEGs identified in tumor samples compared to normal controls, to focus on RNA editing effects on the aberrantly expressed genes in cancers, especially the TGs. F. The overlapping DEGs in the KIRC cancer type. G. The overlapping DEGs were enriched in the immune- and replication-related functions and processes. The RNA editing effects on gene expression in other cancer types are presented in Figures S10–S13. FC, fold change; DEG, differentially expressed gene.

Figure 4

The effects of A-to-I RNA editing events on protein recoding A. There are 3785 non-synonymous and 121 stop-loss editing events causing the changes of amino acid sequences. B. 113 A-to-I RNA editing events conferred their deleterious effects on 52 tumor-related proteins assessed by SIFT, Polyphen2, and PROVEAN. Among these, there were 12 proteins with different RNA editing events whose effects were predicted to be diverse by these three tools. C. The Q/R editing in the key YXXQ motif of the BLCAP protein reverses the inhibition ability of BLCAP to STAT3, potentially facilitating the cancer-initiating and progressing events. D. The hypothesis in (C) was supported by its higher editing frequency in breast invasive carcinoma.

Figure 5

The effects of A-to-I RNA editing events on alternative splicing A. The distribution of altered splice site strength caused by RNA editing events in the different positions of splicing regions. Individual RNA editing site may belong to different groups according to different exons. B. The analysis procedure for the effects of A-to-I RNA editing events on alternative splicing. C. The intron retention event (IR_105891) was mostly occurred in the RNA-edited group of PCPG. D. The intron retention event (IR_105891) was associated with the frequency of the editing event (CAediting_390714) in PCPG. E. A hypothesis that the R/G editing in GRIA2 may alter the canonical splicing pattern of AG-GU to induce the intron retention for the isoforms of tumor-related GRIA2 in PCPG. 3′-ss, 3′-acceptor splice site; 5′-ss, 5′-donor splice site; PSI, percent spliced in; PCPG, pheochromocytoma and paraganglioma.

Figure 6

The effects of A-to-I RNA editing events on miRNA regulation A. RNA editing events in the 3′-UTRs of mRNAs, lncRNAs, and miRNA seed regions led to the changes of miRNA–target interactions. Individual RNA editing site may locate in both of protein-coding or non-coding transcripts due to their possible overlaps. B. The analysis procedure for the effects of A-to-I RNA editing events on miRNA regulation. C. One significant RNA editing event (CAediting_1478179) in the 3′-UTR of APOL1 likely intervened in the miRNA regulation on two TGs (APOL1 and ZNF280D) to play its roles in the UCEC progression. D. The RNA editing event (CAediting_1478179) caused the loss of original miRNA binding target on APOL1. The altered miRNA regulation resulted in the increased expression of APOL1 and indirectly led to the reduced expression of competing ZNF280D gene. E.APOL1 and ZNF280D showed differential expression in UCEC, revealing their potential roles and functions in UCEC. F. Analyses uncovered CAediting_1478179 in the 3′-UTR of APOL1 as a probably pathological biomarker of UCEC. Another significant case shown in panel B is described in Figure S14. UCEC, uterine corpus endometrial carcinoma.