Circular RNA_0000326 promotes bladder cancer progression via microRNA-338-3p/ETS Proto-Oncogene 1/phosphoinositide-3 kinase/Akt pathway

Figures & data

Table 1. Correlation between circ_0000326 expression and clinicopathological parameters

Parameter	n	circ_0000326		P
		High (n = 35)	Low (n = 35)
Age (years)
≥60	30	13	17	0.336
< 60	40	22	18
Gender
Male	36	21	15	0.152
Female	34	14	20
Tumor size (cm)
≥3	40	25	15	0.015
<3	30	10	20
Clinical stage
III-IV	34	22	12	0.016
I-II	36	13	23
Lymphatic metastasis
Negative	34	18	16	0.63
Positive	36	17	19

Figure 1. Circ_0000326 expression was significantly up-regulated in BC. (a). The volcano plot of differentially expressed genes (DECs) in GSE92675. The blue and purple dots represented the downregulated DECs and upregulated DECs with statistical significance (log2 |fold change|>2 and P < 0.05), respectively. (b). The heatmap of DECs whose expressions were upregulated in GSE92675.C-D. qRT-PCR showed that circ_0000326 expression was upregulated in BC tissues and cell lines.***P < 0.001

Figure 2. Circ_0000326 promoted BC cell proliferation and migration, and inhibited the apoptosis. (a). qRT-PCR showed that linear MALAT1 was degraded by RNase R, while circ_0000326 could not be degraded by RNase R. (b). qRT-PCR showed that circ_0000326 expression was inhibited in BC cells transfected with circ_0000326 siRNAs. (c-d). CCK-8 assay showed that cell proliferation in the si-circ_0000326#1 and si-circ_0000326#3 groups was dramatically suppressed as against the si-NC group. (e). Would healing assay indicated that in comparison to the control group, cell migration capacity in the si-circ_0000326#1 and si-circ_0000326#3 groups was significantly reduced. (f). Flow cytometry showed that the apoptosis rate was significantly increased after circ_0000326 knockdown.*P < 0.05, **P < 0.01 and ***P < 0.001

Figure 3. Circ_0000326 could combine with miR-338-3p in BC. (a-b). qRT-PCR indicated that circ_0000326 was mainly distributed in the cytoplasm of 5637 and T24 cell lines. (c). StarBase database was utilized to predict the binding site between circ_0000326 and miR-338-3p. (d). Dual-luciferase reporter assays showed that the luciferase activity of wild-type circ_0000326 reporter was significantly suppressed by miR-338-3p mimics, while the luciferase activity of the mutated reporter was not affected by miR-338-3p mimics. (e). RIP assay showed that circ_0000326 and miR‐338-3p were mainly enriched by anti-Ago2 antibody. (f-g). qRT-PCR showed that miR-338-3p expression was markedly down-regulatedin BC cell lines and tissues. (h). qRT-PCR showed that miR-338-3p expression was significantly increased in BC cells transfected with circ_0000326 siRNA.**P < 0.01 and ***P < 0.001

Figure 4. Circ_0000326 could target miR-338-3p to regulate ETS1 expression

(a). Venn diagram of mRNAs containing the potential binding site to miR-338-3p by searching TargetScan, PicTar, PITA miRmap, microT and miRDB databases. (b). StarBase database was employed to predict the binding site between miR-338-3p and ETS1. (c). Luciferase reporter assay confirmed that miR-338-3p could negatively regulate the luciferase activity of circ_0000326-WT, rather than circ_0000326-Mut. (d). RIP assay showed that miR-338-3p and ETS1 were all enriched in Anti-Ago2 group compared with Anti-IgG group. (e-g). qRT-PCR and Western blot showed that ETS1 expression was notably up-regulatedin BC tissues and cells. (h). qRT-PCR and Western blot showed that miR-338-3p inhibitor counteracted the inhibitory effect of circ_0000326 knockdown on ETS1 expression. (i-k). Pearson correlation analysis was used to analyze the relationships between circ_0000326 and miR-338-3p, miR-338-3p and ETS1 mRNA, as well as miR-338-3p and ETS1 mRNA in BC.**P < 0.01 and ***P < 0.001.

Figure 5. Circ_0000326 could regulate miR-338-3p/ETS1 to participate in BC progression. (a-b). CCK-8 assay was used to verify the cell proliferation in BC cells transfected with si-circ_0000326, circ_0000326 siRNA and miR-338-3p inhibitor, or circ_0000326 siRNA and ETS1 overexpression plasmid. (c-d). Wound healing assay was used to detect the migration of BC cells transfected with si-circ_0000326, circ_0000326 siRNA and miR-338-3p inhibitor, or circ_0000326 siRNA and ETS1 overexpression plasmid. (e-f). Flow cytometry was used to detect the apoptosis of BC cells transfected with si-circ_0000326, circ_0000326 siRNA and miR-338-3p inhibitor, or circ_0000326 siRNA and ETS1 overexpression plasmid. *P < 0.05, **P < 0.01 and ***P < 0.001

Figure 6. Circ_0000326/miR-338-3p/ETS1 activated PI3K/AKT signal pathway in BC

(a-b): GO and KEGG pathway enrichment analyses were used to predict the biological function of ETS1 in BC. (c): Western blot was used to detect p-PI3K, p-AKT, PI3K and AKT protein expressions in BC cells transfected with si-circ_0000326, circ_0000326 siRNA and miR-338-3p inhibitor, or circ_0000326 siRNA and ETS1 overexpression plasmid.