Hsa_circ_0001615 downregulation inhibits esophageal cancer development through miR-142-5p/β-catenin

Abstract

Background: Recent studies have found that circular RNAs (circRNAs) play important roles in tumorigenesis. This study aimed to determine the function and potential mechanisms of hsa_circ_0001615 in esophageal cancer.

Methods: Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) was used to validate the expression of hsa_circ_0001615 and miR-142-5p. Subsequently, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt, flow cytometry, clone formation, and transwell assays were used to assess the function of hsa_circ_0001615. Furthermore, qRT-PCR and Western blot analysis were used to verify cyclin D1, Bcl-2 associated X, B-cell lymphoma/leukemia gene-2, and β-catenin levels. Circular RNA Interactome was used to estimate the binding site between hsa_circ_0001615 and miR-142-5p. Additionally, dual-luciferase reporter assays were used to determine whether miR-142-5p was a direct target of hsa_circ_0001615. Pearson correlation analysis was used to explore the relationship between miR-142-5p and hsa_circ_0001615.

Results: In esophageal cancer, the expressions of hsa_circ_0001615 and miR-142-5p were increased and decreased, respectively. Hsa_circ_0001615 inhibition significantly reduced the proliferation, migration, and invasion but increased the apoptosis of esophageal cancer cells. Additionally, hsa_circ_0001615 knockdown increased miR-142-5p expression but decreased β-catenin expression. MiR-142-5p was a direct target of hsa_circ_0001615.

Conclusion: Hsa_circ_0001615 knockdown could mediate antitumor effects through the miR-142-5p/β-catenin pathway.

Keywords: Esophageal cancer; Hsa_circ_0001615; miR-142-5p; β-catenin.

Figure 1. Hsa_circ_0001615 is elevated in esophageal cancer.

(A) RNase R treatment of PCR amplicons from convergent primers and divergent primers of hsa_circ_0001615, illuminating circularity of hsa_circ_0001615; (B) Sanger sequencing of PCR amplicons from divergent primers confirming the backsplice junction of hsa_circ_0001615; (C) qRT-PCR detection of hsa_circ_0001615 in cancer tissue and adjoining noncancerous tissue. **P < 0.01.

Figure 2. Reduced cell proliferation in hsa_circ_0001615 knockdown cells.

(A) qRT-PCR detection of hsa_circ_0001615 in esophageal cells HEEC, TE-1, ECA-109, and KYSE520; (B) siRNA interfering hsa_circ_0001615 expression markedly repressed hsa_circ_0001615 expression in ECA-109 and KYSE520 cells; (C) MTS assays showed a decreased of cell proliferation in hsa_circ_0001615 knockdown ECA-109 and KYSE520; (D) clone formation assay showed a decreased of clone formation in hsa_circ_0001615 knockdown ECA-109 and KYSE520. ns (not statistically significant), **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Figure 3. Promoted apoptosis in hsa_circ_0001615 knockdown cells.

(A) Expressions of Bax, Bcl2, and Cyclin D1 in ECA-109 and KYSE520 cells detected by qRT-PCR, before and after hsa_circ_0001615 was knockdown; (B) expressions of Bcl2, Cyclin D1, and Bax in in ECA-109 and KYSE-520 cells detected by Western blot, before and after hsa_circ_0001615 was knockdown; (C) flow cytometry results showed increased in apoptosis when hsa_circ_0001615 was knocked down. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Figure 4. Migration and Invasion of ECA-109 and KYSE520 cells after hsa_circ_0001615 was Knockdown.

(A) Transwell assays in ECA-109 with hsa_circ_0001615 knockdown showed a decreased number of migration and invasion cells; (B) transwell assays in KYSE-520 with hsa_circ_0001615 knockdown showed a decreased number of migration and invation cells. **P < 0.01, and ***P < 0.001.

Figure 5. Hsa_circ_0001615 regulates miR-142-5p through direct binding.

(A) All miRNAs that were predicted to have a targeting relationship with hsa_circ_0001615; (B) Dual luciferase reporter assay to detect the binding site between hsa_circ_0001615 and miR-142-5p; (C) qRT-PCR detection of miR-142-5p in cancer tissue and adjoining noncancerous tissue (n = 30); (D) relationship between hsa_circ_0001615 and miR-142-5p in esophageal tumor tissue (n = 30); (E) miR-142-5p expression detected by qRT-PCR in KYSE520 and ECA-109 cells, before and after hsa_circ_0001615 was knockdown; (F) expressions of β-catenin in KYSE520 and ECA-109 cells detected by Western blot, before and after hsa_circ_0001615 was knockdown. ns (not statistically significant), *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.