Expression profiling of circular RNA reveals a potential miR-145-5p sponge function of circ-AFF2 and circ-ASAP1 in renal cell carcinoma

Abstract

Objectives: Circular RNAs (circRNAs) are involved in carcinogenesis, though their expression profile in renal cell carcinoma (RCC) is uncharacterized. The tumor suppressor gene miR-145-5p is expressed in RCC tissues, but its relationship with circRNAs is unknown. Thus, we aimed to identify differentially expressed circRNAs in RCC tissues and to explore the interaction between these circRNAs and miR-145 in the development of RCC.

Methods: We performed high-throughput sequencing and bioinformatics analyses to examine the expression pattern of circRNAs in RCC. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were used to functionally annotate differentially expressed circRNAs. Quantitative real-time polymerase chain reaction (qRT-PCR) was used for sequence verification. Small interfering RNAs were employed to investigate the function and mechanism of circRNAs in RCC. The relationship between miR-145-5p and circRNAs was confirmed using luciferase, RNA immunoprecipitation (RIP), and biotin-coupled probe RNA pull-down assays.

Results: Fifty-three circRNAs were significantly and differentially expressed in RCC compared to normal control tissue. Bioinformatic analyses indicated that two significantly upregulated circRNAs, circ-AFF2 and circ-ASAP1, had sequences corresponding to miR-145 response elements. Consistently, the luciferase reporter, RIP, and biotin-coupled probe RNA pull-down assays showed that circ-AFF2 and circ-ASAP1 may repress miR-145 by acting as sponges. circ-AFF2 and circ-ASAP1 were highly expressed in RCC patient-derived tumor samples; their overexpression correlated with poor prognosis and low miR-145 levels. Knockdown of circ-AFF2 or circ-ASAP1 in RCC cell lines inhibited proliferation, underscoring their oncogenic function. A circRNA-miRNA network was constructed for RCC using the differentially expressed circRNAs and projected miRNAs. Candidate genes were verified by RT-qPCR and western blot, indicating that circ-AFF2 and circ-ASAP1 may be connected to RCC proliferation and metastasis.

Conclusion: circ-AFF2 and circ-ASAP1 were upregulated in RCC and likely promote tumor progression by sponging miR-145. Therefore, both circRNAs should be investigated further as potential diagnostic and therapeutic targets for RCC.

Keywords: Circular RNA; circ-AFF2; circ-ASAP1; high-throughput sequencing; miR-145-5p; renal cell carcinoma.

Figure 1

Circular RNA (circRNA) profile of renal cell carcinoma (RCC). A. The proportion of novel circRNAs among the identified circRNAs in RCC. B. Classification of circRNAs. C. Chromosomal distribution of circRNAs. D. circRNA length distribution. E. circRNA distribution per gene. F. Venn diagram illustrating differentially expressed circRNAs. G. Novel circRNA distribution in differentially expressed circRNAs.

Figure 2

Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses of differentially expressed circRNAs. GO annotation of the differentially expressed circRNAs for (A) molecular function, (B) cellular components, and (C) biological process. The bar plots illustrate the top ten enriched terms of each category. (D) KEGG pathways of the differentially expressed circRNAs. The horizontal axis represents the RichFactor and the vertical axis represents the pathway name.

Figure 3

Characteristics and schematic illustration of circ-AFF2. A. Annotation of the predicted binding site sequence between circ-AFF2 and miR-145. The “2D Structure” column displays the binding sequence of circ-AFF2 and miR-145. The “Local AU” column shows the upstream and downstream sequences of the seed sequence consisting of 30 nucleotides. The “Position” column illustrates the possible position of miRNA response elements on the circRNA sequence. B. Characteristics and schematic illustration of circ-AFF2. Box 1-GC percentage and evolutionary conservation of AFF2 genes. Box 2-Specific back-splice site of circ-AFF2 as verified by Sanger sequencing.

Figure 4

Characteristics and schematic illustration of circ-ASAP1. A. Annotation of the predicted binding site sequence between circ-ASAP1 and miR-145. B. Features and schematic illustration of circ-ASAP1. Box 1-GC percentage and evolutionary conservation of ASAP1 genes. Box 2-Specific back-splice site of circ-ASAP1 as verified by Sanger sequencing.

Figure 5

circ-AFF2 and circ-ASAP1 partially inhibit the proliferation of RCC cells in response to miR-145-5p. A, B. Relative expression levels of circ-AFF2, AFF2 mRNA, circ-ASAP1, and ASAP1 mRNA in ACHN cells after RNase R treatment. C, D. RCC cell growth patterns following transfection with si-circ-AFF2 or si-circ-ASAP1. E, F. RCC cell growth patterns after transfection with negative control (NC), si-circ, or si-circ+miR-inhibitor. *P<0.05, **P<0.001.

Figure 6

circ-AFF2 and circ-ASAP1 serve as sponges for miR-145-5p in RCC cell lines. A. Relative luciferase activity in RCC samples transfected with circ-AFF2-WT or circ-AFF2-Mut and miR-NC (control) or miR-145-5p mimic; wild-type and mutant sequences of the circ-AFF2 luciferase vectors. B. Relative luciferase activity in RCC samples transfected with circ-ASAP1-WT or circ-ASAP1-Mut and miR-NC or miR-145-5p mimic; wild-type and mutant sequences of the circ-ASAP1 luciferase vectors. C. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to determine the expression levels of miR-145-5p in RCC cells after transfection with miR-145 mimics, miR-145 inhibitor, si-circ-AFF2, si-circ-ASAP1, or controls. D. After miR-145 mimics and NC mimics were transfected into ACHN cells, anti-Ago2 RNA immunoprecipitation (RIP) assays were performed to measure circ-AFF2 and circ-ASAP1 expression levels. E. Using circ-AFF2 or circ-ASAP1-specific probes, miR-145 was recovered. *P<0.05, **P<0.001.

Figure 7

circ-AFF2 and circ-ASAP1 regulatory mechanisms in RCC. A. The circRNA-miRNA-mRNA interaction network. Both hsa_circ_0001947 (circ-AFF2) and hsa_circ_0008934 (circ-ASAP1) are represented by yellow circles. Green arrowheads represent the top nine miRNAs; target mRNAs are presented as red octagons. B. Various proliferation-related gene expression levels were examined by qRT-PCR in ACHN cells transfected with NC, si-circ-AFF2, or si-circ-ASAP1. C. Western blot analysis was used to determine the protein levels of ABCE1, SRGAP2, and FSCN1 in ACHN cells transfected with NC, si-circ-AFF2, or si-circ-ASAP1. *P<0.05, **P<0.001.

Figure 8

Clinical validation of circ-AFF2 and circ-ASAP1 in RCC specimens. Expression levels of circ-AFF2 (A), circ-ASAP1 (B), and miR-145 (C) in 31 RCC tissue samples relative to adjacent normal control samples. (D and E) The receiver operating characteristic (ROC) curve of circ-AFF2 (D) and circ-ASAP1 (E) in distinguishing RCC. AUC: Area under the Curve. *P<0.05, **P<0.001.