CircRNA_0000392 promotes colorectal cancer progression through the miR-193a-5p/PIK3R3/AKT axis

Abstract

Background: Circular RNAs (circRNAs), important members of the noncoding RNA family, have been recently revealed to play a role in the pathogenic progression of diseases, particularly in the malignant progression of cancer. With the application of high-throughput sequencing technology, a large number of circRNAs have been identified in tumor tissues, and some circRNAs have been demonstrated to act as oncogenes. In this study, we analyzed the circRNA expression profile in colorectal cancer (CRC) tissues and normal adjacent tissues by high-throughput sequencing. We focused on circRNA_0000392, a circRNA with significantly increased expression in CRCtissues, and further investigated its function in the progression of colorectal cancer.

Methods: The expression profile of circRNAs in 6 pairs of CRC tissues and normal adjacent tissues was analyzed by RNA sequencing. We verified the identified differentially expressed circRNAs in additional samples by qRT-PCR and selected circRNA_0000392 to evaluate its associations with clinicopathological features. Then, we knocked down circRNA_0000392 in CRC cells and investigated the in vitro and in vivo effects using functional experiments. Dual luciferase and RNA pull-down assays were performed to further explore the downstream potential molecular mechanisms.

Results: CircRNA_0000392 was significantly upregulated in CRC compared with normal adjacent tissues and cell lines. The expression level of circRNA_0000392 was positively correlated with the malignant progression of CRC. Functional studies revealed that reducing the expression of circRNA_0000392 could inhibit the proliferation and invasion of CRC both in vitro and in vivo. Mechanistically, circRNA_0000392 could act as a sponge of miR-193a-5p and regulate the expression of PIK3R3, affecting the activation of the AKT-mTOR pathway in CRC cells.

Conclusions: CircRNA_0000392 functions as an oncogene through the miR-193a-5p/PIK3R3/Akt axis in CRC cells, suggesting that circRNA_0000392 is a potential therapeutic target for the treatment of colorectal cancer and a predictive marker for CRC patients.

Keywords: CircRNA_0000392; Circular RNAs (circRNAs); Colorectal cancer; PIK3R3; RNA sequencing; miR-193a-5p.

Fig. 1

Identification of circular RNAs by RNA-seq analyses in human CRC samples. a The scatter plot shows the changes in circRNA expression in six paired CRC and adjacent normal tissues (ANT). CircRNAs above the top green line and below the bottom green line demonstrated a greater than 1.5-fold change between the two compared groups. b The volcano plot shows the expression profiling of circRNA between CRC and ANT. The vertical green lines refer to a 2.0-fold (log2 scaled) upregulation and downregulation, respectively. The horizontal green line corresponds to a P-value of 0.05 (−log10 scaled). The red points in the plot represent differentially expressed circRNAs with statistical significance. c Clustered heat map indicating differences in circRNA expression profiling between CRC and ANT tissues. d The number of total circRNAs identified by RNA-seq and the number of differentially expressed circRNAs. e CircRNAs were classified by categories. f Validation of the top 4 differentially expressed circRNAs in 16 paired CRC and ANT tissues by RT-qPCR. CRC, colorectal cancer; ANT, adjacent normal tissue. Data represent the mean ± SD. * P < 0.05, ** P < 0.01

Fig. 2

CircRNA_0000392 is upregulated in CRC and associated with progression. a - b Relative expression of circRNA_0000392 in 40 pairs of CRC and ANT tissues measured by qRT-PCR. c ROC curve analysis of circRNA_0000392 in CRC patients. AUC values are included in the graphs. d Percentages of CRC tissues with low or high expression of circRNA_0000392 according to TNM stage. e Analysis of circRNA_0000392 expression in CRC patients according to TNM stage. f Analysis of circRNA_0000392 expression in CRC patients with or without lymph node metastasis. g Analysis of circRNA_0000392 expression in CRC patients with or without distant metastasis. h Relative expression of circRNA_0000392 in cell lines was detected by qRT-PCR. Data represent the mean ± SD. * P < 0.05, ** P < 0.01

Fig. 3

The characteristics of circRNA_0000392. a The genomic loci of the YAF2 gene and circRNA_0000392. The spliced mature sequence length of circRNA_0000392 is 326 bp. b The expression of circRNA_0000392 and YAF2 mRNA in SW620 and RKO cells treated with or without RNase R was detected by qRT-PCR. c qRT-PCR analysis of circRNA_0000392 and YAF2 mRNA in SW620 cells treated with actinomycin D at the indicated time points. d Fluorescence in situ hybridization (FISH) assays were performed to determine the localization of circRNA_0000392 in SW620 and RKO cells. Scale bar = 50 μm. Data represent the mean ± SD. * P < 0.05, ** P < 0.01

Fig. 4

Knockdown of circRNA_0000392 inhibits CRC cell proliferation and invasion. a qRT-PCR analysis of circRNA_0000392 in SW620 and RKO cells after transfection with siRNA for 48 h. b - c SW620 and RKO cell proliferation after circRNA_0000392 knockdown by siRNA was detected by WST-1 assay. d The apoptosis rate was analyzed by flow cytometry after downregulation of circRNA_0000392 in SW620 and RKO cells. (E - F) Cell migration (e) and invasion (f) were assessed by transwell assay with or without Matrigel after circRNA_0000392 knockdown in SW620 and RKO cells. Data represent the mean ± SD. * P < 0.05, ** P < 0.01

Fig. 5

CircRNA_0000392 functions as a sponge for miR-193a-5p. a RIP analysis of circRNA_0000392 using anti-AGO2 antibody in SW620 and RKO cells. b The circRNA-miRNA-mRNA interaction based on circRNA_0000392 was demonstrated by prediction and bioinformatics analysis using Cytoscape software. c The top five miRNAs that may be regulated by circRNA_0000392 based on the miRNA prediction and bioinformatics analyses are shown and measured by qRT-PCR after the pull-down assay in RKO cells. d Schematic illustration demonstrating the luciferase reporter vectors containing wild-type (WT) or mutant (MUT) predicted miR-193a-5p binding sites of circRNA_0000392. e The luciferase assay was performed in 293 T cells after cotransfection with miR-193a-5p mimic and the luciferase vector containing wild-type (WT) or mutant (MUT) circRNA_0000392. f Relative expression of miR-193a-5p in 40 pairs of CRC and ANT tissues measured by qRT-PCR. g The correlation between circRNA_0000392 and miR-193a-5p in CRC tissues was analyzed by Spearman correlation coefficients. Data represent the mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001

Fig. 6

PIK3R3 is directly targeted by miR-193a-5p and indirectly regulated by circRNA_0000392. a The relative mRNA expression of EPHA2, PIK3R3, EGFR, USP22 and DDX58 after transfection with the miR-193a-5p inhibitor was detected in SW620 cells by qRT-PCR. b Schematic illustration of PIK3R3 3’UTR wild-type (WT) or 3’UTR mutant (MUT) luciferase reporter vectors and the predicted binding sites to miR-193a-5p. c The relative luciferase activities were detected in 293 T cells after cotransfection with the PIK3R3 3’UTR wild-type (WT) or 3’UTR mutant (MUT) luciferase reporter vectors with the miR-193a-5p mimics. d Relative PIK3R3 mRNA expression after transfection with the miR-193a-5p mimics or inhibitor was detected in cells by qRT-PCR. e The relative PIK3R3 protein level after transfection with the miR-193a-5p mimics was detected in cells by western blot. f Relative expression of PIK3R3 in 40 pairs of CRC and ANT tissues measured by qRT-PCR. g Relative PIK3R3 mRNA expression after transfection with circRNA_0000392 siRNA was detected by qRT-PCR. h Representative IHC staining images of low and high PIK3R3 expression in patient CRC tissue samples. Scale bar = 20 μm. i The correlation between circRNA_0000392 and PIK3R3 protein expression in CRC tissues was analyzed based on Spearman correlation coefficients. Data represent the mean ± SD. * P < 0.05, ** P < 0.01

Fig. 7

CircRNA_0000392 promotes cell proliferation and invasion through the circRNA_0000392/miR-193a-5p/PIK3R3 axis. a - b SW620 and RKO cell proliferation after transfection with circRNA_0000392 siRNA and/or miR-193a-5p inhibitor was measured by WST-1. c - d The cell migration (c) and invasion (d) capabilities were determined by Transwell assay after transfection of SW620 and RKO cells with the circRNA_0000392 siRNA and/or miR-193a-5p inhibitor. e The relative mRNA expression of PIK3R3 after transfection with circRNA_0000392 siRNA and/or miR-193a-5p inhibitor was detected by qRT-PCR. f - i The relative protein expression of PIK3R3 and the phosphorylation level of downstream pathway proteins were measured by western blot in cells transfected with the circRNA_0000392 siRNA and/or miR-193a-5p inhibitor. Data represent the mean ± SD. * P < 0.05, ** P < 0.01

Fig. 8

Downregulation of circRNA_0000392 suppresses the growth of CRC cells in vivo. a Image of subcutaneous xenograft tumors. Nude mice were injected with 5 × 10⁶ SW620 cells (n = 5 for each group). Tumors were extracted after 30 days. b Analysis of tumor volume of mice measured every 3 days. c Tumor weight in each group at the end of the experiment. d Histological analysis of tumor tissues by hematoxylin and eosin staining. IHC of Ki-67 and PIK3R3 in subcutaneous tumors. Scale bar, 100 μm. e The graph shows the relative signal intensity scores of KI-67 and PIK3R3. f Schematic illustration of circRNA_0000392 regulating the miR-193a-5p/PIK3R3 axis in CRC. Data represent the mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001. Additional file 1