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

Abstract

Circular RNAs (circRNAs) play a pivotal regulatory role in bladder cancer (BC) occurrence and progression. The expression level, role and mechanism of circ_0000326 in BC remain unknown. In the present study, quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was conducted to evaluate the expressions of circ_0000326, microRNA-338-3p (miR-338-3p) and ETS Proto-Oncogene 1(ETS1) mRNA in BC tissues and cell lines. Cell counting kit-8 (CCK-8) assay, wound healing assay and flow cytometry were used to detect the impacts of circ_0000326 on BC cell growth, migration and apoptosis. Western blot was used to detect the expressions of ETS1, phospho-phosphoinositide-3 kinase (p-PI3K), phospho-AKT, PI3K and AKT protein. Gene ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed to analyze the biological function of ETS1 in BC. Here, we found that circ_0000326 expression was significantly elevated in BC cell lines and tissues, and circ_0000326 could promote BC cell growth and migration, and inhibit apoptosis. Dual-luciferase reporter gene assay confirmed that circ_0000326 and ETS1 could bind directly to miR-338-3p. Furthermore, circ_0000326 sponged miR-338-3p and up-regulated ETS1 expression. ETS1 was associated with the activation of PI3K/AKT pathway. Moreover, circ_0000326 could activate PI3K/AKT pathway by miR-338-3p/ETS1 axis. Collectively, circ_0000326/miR-338-3p/ETS1/PI3K/AKT pathway is involved in regulating BC progression.

Keywords: Circ_0000326; ETS1; PI3K/AKT; bladder cancer; miR-338-3p.

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

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