Thousands of genes are deregulated upon circHIPK3 knockdown

(A) circHIPK3 is produced from the second exon of the HIPK3 gene. We designed a circHIPK3-specific siRNA targeting the backsplice junction of circHIPK3 to specifically knockdown the expression of circHIPK3. (B) Expression of circHIPK3 and the corresponding linear transcript in eleven bladder cancer cell lines. UMUC3 and J82 were chosen for knockdown experiments based on circHIPK3 expression levels and cell line stability. CPM = counts per million. (C) Knockdown efficiency of circHIPK3 in UMUC3 and J82 at different time points post circHIPK3 (yellow) or scramble (grey) siRNA transfection. Expression is normalized to GAPDH levels. (D) PCA plot of gene expression in UMUC3 and J82. PCA plots are based on the genes with the 50% most variance across all cell line samples. Gene expression is log transformed (natural logarithm) and added a pseudocount of 1. (E) Overlap of differentially expressed genes across all timepoints and/or conditions between UMUC3 (n = 3,072) and J82 (n = 2,389). P-value obtained by Fisher’s Exact Test. (F) Number of differentially expressed genes at each time point (Scr vs. circHIPK3) (Wald test, Benjamini-Hochberg correction with FDR < 0.1). Only genes with perturbed expression profiles across time and/or conditions in UMUC3 (n = 3,072) or J82 (n = 2,389) are considered. (G) Differential expression analysis between circHIPK3 KD and scr siRNA samples 24 hour post-transfection (Wald test). The log2 fold changes (circHIPK3 KD vs scr) are plotted against the negative log10(p-values). Colors indicate if genes are significantly down-(red) or upregulated (blue) or not differentially expressed (Not DE, green) after Benjamini-Hochberg correction, FDR < 0.1. Vertical lines indicate a log2FC > 1 or < - 1.

A long motif in circHIPK3 is enriched in downregulated genes upon circHIPK3 knockdown

(A) Illustration of conserved miRNA binding sites in circHIPK3 based on AGO-CLIP data. (B) Percentage of genes in each group with miR-148ab-3p/152 target sites (CAGUGCA) in their 3’UTRs. Gene regulation is based on circHIPK3 KD vs scr siRNA transfection 24 hours (24h) post-transfection in J82. P-values obtained by Chi-square Test. (C) Procedure for motif enrichment analysis using Regmex. First, we extract all 7-mers in the sequence of circHIPK3. For each 7-mer, e.g., CCCTGCC, we identify their presence in the 3’UTR of genes. We order all genes according to a differential expression score calculated as the -log10(P-value) multiplied by the fold change direction, e.g., 1 for upregulated genes and −1 for downregulated genes. Then we calculate a motif score for each 7-mer based on their occurrences in either down- or upregulated genes. If a 7-mer has a positive motif score it means that it is enriched in the 3’UTR of genes that are upregulated upon circHIPK3 KD. Conversely, 7-mers with a negative motif score are primarily found in genes that are downregulated upon circHIPK3 KD. The 7-mer, CCCTGCC, is used for illustration purposes. (D) Regmex motif scores for circHIPK3 7-mers (UMUC3, 24h). Alignment of the ten 7-mers with the most negative motif scores are shown. 7-mers are ranked from most negative to most positive motif scores. Numbers correspond to rank. (E) Illustration of circHIPK3 and position of the ten motifs with the lowest motif scores. The 7-mers with the most negative motif scores found in circHIPK3 comprise a larger 11-mer, AGGCCCCCAGC, present in the sequence of circHIPK3. (F) Percentage of genes in each group containing the 11-mer motif upon circHIPK3 KD in UMUC3 cells (24h). P-values obtained by Chi-square Test.

The 11-mer motif in circHIPK3 constitutes a binding site for IGF2BP2

(A) Illustration of RBP binding sites in circHIPK3 based on eCLIP data in the ENCODE cell lines HepG2 and K562. (B) RNA immunoprecipitation of circHIPK3-RBPs and others in HepG2 and K562 cells. Bands semi-quantitatively confirm that circHIPK3 interacts with IGF2BP1 and IGF2BP2. Marker indicates 50 bp. (C) Percentage of genes in each group containing IGF2BP2 binding sites (K562) in UMUC3 (24h). P-values obtained by Chi-square Test. (D) Overlap between genes that are affected by circHIPK3 KD in UMUC3 and IGF2BP2 KD in K562. P-value obtained by Fisher’s Exact Test. (E) Percentage of genes in each group affected by IGF2BP2 KD in K562. P-values obtained by Chi-square Test. (F) Regulation of genes affected by both circHIPK3 KD (UMUC3, 24h) and IGF2BP2 KD (K562). The x-axis indicates gene regulation upon circHIPK3 KD in UMUC3. Percentage on y-axis and colors show how these genes are regulated upon IGF2BP2 KD. Downregulated genes upon circHIPK3 KD are mainly upregulated upon IGF2BP2 KD and vice versa. (G) Regmex motif enrichment analysis upon IGF2BP2 KD in K562. 7-mers are ranked from most positive to most negative motif scores. The ten 7-mers with the most negative motif scores in UMUC3 are shown. Numbers indicate motif rank. All possible 7-mers are evaluated (n = 16,384). (H-I) Percentage of genes in each group containing the 11-mer motif (H) and IGF2BP2 binding sites (I) upon IGF2BP2 KD in K562. P-values obtained by Chi-square Test.

circHIPK3 interacts with IGF2BP2 and affects genes controlling cell cycle progression

(A) Relative enrichment of circHIPK3 levels or ACTB mRNA between IP and input for the three RBPs GRWD1, IGF2BP1, and IGF2BP2 in the bladder cancer cell line FL3. GFP was used as a negative control. The statistical difference in circHIPK3 enrichment for GRWD1 and IGF2BP1 or IGF2BP2 is indicated by the P-value (T-Test). (B) Relative enrichment of circHIPK3 levels between IP and input for IGF2BP2 upon circHIPK3 overexpression (OE – 24 hrs). GFP was used as a negative control. Error bars reflect standard deviation of biological triplicates. (C) Percentage of genes in each group containing the 11-mer motif upon IGF2BP2 KD in UMUC3. P-values obtained by Chi-square Test. (D) Gene set enrichment analysis of 50 hallmarks of cancer upon circHIPK3 KD (24h) in UMUC3 cells (FDR < 0.1 for all shown pathways). (E) Downregulated hallmarks of cancer upon IGF2BP2 KD in UMUC3 (P-value < 0.05 for all shown pathways).

circHIPK3 functions as a competing endogenous RNA for IGF2BP2

(A) STAT3 mRNA expression upon circHIPK3 KD in the time-course perturbation experiment in UMUC3 cells. Expression represents DESeq2 normalized counts. P-value (24h) obtained by Wald Test. (B) Expression of STAT3 upon IGF2BP2 knockdown in UMUC3 cells. P-value obtained by Wald Test. (C) Relative enrichment of STAT3 and β-actin levels between IGF2BP2 IP and input in FL3 cells. GFP was used as a negative control. P-value obtained by T-test. (D) Relative enrichment of STAT3 levels between IGF2BP2 IP and input upon circHIPK3 KD in FL3 cells. P-value obtained by T-test. Control sample (scr) has been normalized to 1. (E) Relative enrichment of STAT3 and GAPDH levels between IGF2BP2 IP and input after 11-mer ASO transfection in FL3 cells. P-values obtained by T-test. (F) Relative enrichment of circHIPK3, STAT3, and GAPDH levels between IGF2BP2 IP and input after transfection of wildtype and mutated circHIPK3 in HEK293 cells. P-values obtained by T-test. (G) TP53 mRNA expression upon circHIPK3 KD in the time-course perturbation experiment in UMUC3 cells. Expression represents DESeq2 normalized counts. P-value (72h) obtained by Wald Test. (H) Western blot of STAT3 and p53 protein upon circHIPK3 KD in UMUC cells. Quantifications on the left are internally normalized to HuR and then scr is normalized to 1. P-values obtained by T-test. (I-J) Immunofluorescence staining for STAT3 (I) or p53 (J) in fixed UMUC3 cells (as indicated - green) subjected to control (scr) or circHIPK3 KD. Nuclei were counterstained using DAPI (as indicated - blue). Mean signal intensities are quantified within nuclei or entire cells to the right. P-values obtained by Wilcoxon Rank Sum Test. (B-F, H) Error bars reflect standard deviation of biological triplicates.

Absolute quantifications suggest low levels of circHIPK3 compared to IGF2BP2, where high circHIPK3 levels correlate with increased survival of bladder cancer patients

(A) Flow chart used to perform absolute quantification of circHIPK3 and IGF2BP2 protein levels in BC cells. (B) Estimated levels of circHIPK3 and IGF2BP2 (copies per cell). (C) Immunofluorescence using IGF2BP2 antibody (red channel) with cells transfected with circHIPK3 expression plasmid (upper panel) or control plasmid (lower panel). Both plasmids encode enhanced green fluorescent protein (GFP) from a separate expression cassette in order to identify transfected cells (green channel). White arrows indicate IGF2BP2 condensates (D) Quantification of transfected cells containing large IGF2BP2 condensates - % positive cells (28 and 34 cells were counted). Error bars reflect standard error of mean (SE). (E) Expression of circHIPK3 in samples from patients with non-muscle invasive bladder cancer (NMIBC) and muscle invasive bladder cancer (MIBC). CPM = counts per million. P-value obtained by Wilcoxon Rank Sum Test. (F) Kaplan–Meier overall survival plots of circHIPK3 (left) and HIPK3 (right). Median expression of circHIPK3 (0.192 CPM) and HIPK3 (7.96 FPKM) used as cutoff. P-values obtained by Log-Rank Test. (G) Working model that illustrates potential mechanism for circHIPK3. Our studies show that circHIPK3 contains an 11-mer motif (illustrated by red bases) that comprise a binding site for IGF2BP2 (in green). At normal levels, circHIPK3 interacts strongly with IGF2BP2 (middle panel), potentially involving nucleation of many IGF2BP2 proteins on single circHIPK3 molecules - exacerbated at high circHIPK3 levels, which induce condensates (left panel). This allows for only a few accessible IGF2BP2 molecules to bind target mRNAs including STAT3. Upon circHIPK3 knockdown and low circHIPK3 levels (right panel), IGF2BP2 is more accessible to interact more strongly with IGF2BP2 target genes, which contain the 11-mer motif in their 3’UTRs, incl. STAT3, and which are subsequently downregulated. Conversely, upon IGF2BP2 knockdown, IGF2BP2 target genes containing 11-mer are upregulated (not illustrated).