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. 2016 May 3;7(18):26680-91.
doi: 10.18632/oncotarget.8589.

Emerging roles of circRNA_001569 targeting miR-145 in the proliferation and invasion of colorectal cancer

Huijun Xie  1   2 , Xiaoli Ren  1   2 , Sainan Xin  1   2 , Xiaoliang Lan  3 , Guifeng Lu  1   2 , Yuan Lin  1   2 , Shaoshan Yang  1   2 , Zhicheng Zeng  1   2 , Wenting Liao  1   2 , Yan-Qing Ding  1   2 , Li Liang  1   2
Affiliations

Emerging roles of circRNA_001569 targeting miR-145 in the proliferation and invasion of colorectal cancer

Huijun Xie et al. Oncotarget. .

Abstract

Circular RNAs (circRNAs), a large class of RNAs, have recently shown huge capabilities as gene regulators in mammals. Some of them bind with microRNAs (miRNAs) and act as natural miRNA sponges to inhibit related miRNAs' activities. Here we showed that hsa_circ_001569 acted as a positive regulator in cell proliferation and invasion of colorectal cancer (CRC). Moreover, hsa_circ_001569 was identified as a sponge of miR-145 and up-regulated miR-145 functional targets E2F5, BAG4 and FMNL2. In CRC tissues, circ_001569 negatively correlated with miR-145, and miR-145 correlated negatively with E2F5, BAG4 and FMNL2 expressions. Our study reveals a novel regulatory mechanism of circ_001569 in cell proliferation and invasion in CRC, provides a comprehensive landscape of circ_001569 that will facilitate further biomarker discoveries in the progression of CRC.

Keywords: BAG4; FMNL2; colorectal cancer; hsa_circ_001569; miR-145.

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Conflict of interest statement

No potential conflicts of interest were declared.

Figures

Figure 1
Figure 1. Up-regulation of circ_001569 correlates with aggressive characteristics of CRC
(A) Expression of circ_001569 in the 30 paired human CRC tissues and normal intestine epithelial tissues by real-time PCR. Each bar represents the mean of 3 independent experiments. (B) Correlation between circ_001569 expression and T classification (T1–T4) in 30 cases of CRC tissues and normal intestine epithelial tissues. (C) Correlation between circ_001569 expression and N classification of CRC (N0–N2). (D) Correlation between circ_001569 expression and distant metastasis. (E) Correlation between circ_001569 expression and differentiation of CRC. *p < 0.05, **p < 0.01.
Figure 2
Figure 2. Circ_001569 promotes proliferation and tumor growth of CRC cells
(A) Effect of circ_001569 on cell proliferation in vitro by CCK8 assay. (B) Effect of circ_001569 on CRC cell cycle by flow cytometry. (C) Effect of circ_001569 on CRC cell apoptosis by flow cytometry. (D) Effect of circ_001569 on CRC cell invasion in vitro by Boyden chamber. Morphological comparison of cells penetrating the artificial basement membrane was also shown. Scale bars represent 50 μm. Error bars represent mean ± SD from three independent experiments. *p < 0.05, **p < 0.01.
Figure 3
Figure 3. Circ_001569 inhibits the transcription activity of miR-145 and up-regulates miR-145 targets E2F5, BAG4 and FMNL2
(A) Effects of empty vector (EV), circ_001569, circ_001569 inhibitor (circ-in), miR-145 inhibitor (miR-in) and miRNA inhibitor control (IC) on the luciferase activity of miR-145 in SW480 and LOVO cells by luciferase reporter assay. (B) Expression of miR-145 in cells transfected with circ_001569, circ_001569/miR-145, circ_001569 inhibitor (circ-in) and circ-in/miR-145 inhibitor (miR-145-in), respectively. (C) Effect of miR-145 on the luciferase activities of E2F5, BAG4 and FMNL2 3′UTR and their mutant fragments in SW480 and HCT116 cells by luciferase reporter assay. (D) Expressions of E2F5, BAG4 or FMNL2 in miR-145 expressing or depleting cells by Western Blotting and was normalized by β-actin expression. (E) Expressions of E2F5, BAG4 or FMNL2 in circ_001569 expressing or depleting cells by Western Blotting and was normalized by α-tublin expression. Error bars represent mean ±SD from three independent experiments. *p < 0.05, **p < 0.01.
Figure 4
Figure 4. Circ_001569 promotes the proliferation and invasion of CRC cells by regulating miR-145 and its targets
(A) Effect of miR-145 on circ_001569 induced cell proliferation by CCK-8 assay. (B) Effect of miR-145 on circ_001569 induced CRC cell cycle by flow cytometry. (C) Effect of miR-145 on circ_001569 induced CRC cell apoptosis by flow cytometry. (D) Effect of miR-145 on circ_001569 induced cell invasion in vitro by Boyden chamber. (E) Effect of E2F5, BAG4 or FMNL2 on circ_001569 knockdown induced cell proliferation by CCK-8 assay. (F) Effect of E2F5, BAG4 or FMNL2 on circ_001569 knockdown induced CRC cell cycle by flow cytometry. (G) Effect of E2F5, BAG4 or FMNL2 on circ_001569 knockdown induced CRC cell apoptosis by flow cytometry. (H) Effect of E2F5, BAG4 or FMNL2 on circ_001569 knockdown induced cell invasion in vitro by Boyden chamber. Error bars represent mean ± SD from three independent experiments.*p < 0.05, **p < 0.01.
Figure 5
Figure 5. E2F5, BAG4 or FMNL2 plays crucial roles in miR-145 induced the proliferation and invasion of CRC cells
(A) Effect of E2F5, BAG4 or FMNL2 on miR-145 induced cell proliferation by CCK-8 assay. (B) Effect of E2F5, BAG4 or FMNL2 on miR-145 induced CRC cell cycle by flow cytometry. (C) Effect of E2F5, BAG4 or FMNL2 on miR-145 induced CRC cell apoptosis by flow cytometry. (D) Effect of E2F5, BAG4 or FMNL2 on miR-145 induced cell invasion in vitro by Boyden chamber. Error bars represent mean ±SD from three independent experiments.*p < 0.05, **p < 0.01.
Figure 6
Figure 6. MiR-145 negatively correlates with circ_001569 or its targets
(A) Expressions of miR-145 in 30 paired human CRC tissues and the corresponding normal mucosa by real-time PCR. *p < 0.05. (B) Expression correlation between hsa_circ_001569 and miR-145. (C) Expressions of E2F5, BAG4 and FMNL2 in 30 paired human CRC tissues and the corresponding normal mucosa by Western blotting. (D–F) Expression correlations between miR-145 and E2F5, BAG4 or FMNL2.
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