Deregulation of circRNA hsa_circ_0009109 promotes tumor growth and initiates autophagy by sponging miR-544a-3p in gastric cancer

Abstract

Background: Autophagy death of cancer cells is detrimental to apoptosis induced by therapeutic drugs, which promotes tumor progression to a certain extent. Increasing reports have demonstrated the regulatory role of circular RNAs (circRNAs) in autophagy. Here, we aimed to determine the role of hsa_circ_0009109 in autophagy in gastric cancer (GC).

Methods: The effects of hsa_circ_0009109 on autophagy were examined using quantitative real-time polymerase chain reaction (qPCR), transmission electron microscopy, Western blot, and immunofluorescence. The mechanism of hsa_circ_0009109 regulating the miR-544a-3p/bcl-2 axis was analysed using fluorescence in situ hybridization, dual-luciferase reporter, and rescue experiments.

Results: Functional testing indicated that hsa_circ_0009109 was significantly down-expressed in GC tissues and cell lines. A reduction in cytoplasmic-derived hsa_circ_0009109 could promote GC progression by accelerating cell proliferation, enhancing migration and invasion, inhibiting apoptosis, and accelerating the cell cycle progression. Besides, hsa_circ_0009109 was found to exert the effect of an autophagy inhibitor such as 3-Methyladenine (3-MA), which was manifested by the weakening of the immunofluorescence of LC3B and the reduction in autophagy-related proteins after overexpression of hsa_circ_0009109, while increased autophagosomes were observed after interference with hsa_circ_0009109. Subsequently, the crosstalk between hsa_circ_0009109 and miR-544a-3p/bcl-2 was verified using dual-luciferase reporter assay. The autophagy status was altered under the regulation of the hsa_circ_0009109-targeted miR-544a-3p/bcl-2 axis.

Conclusions: The hsa_circ_0009109 mediated a novel autophagy regulatory network through targeting the miR-544a-3p/bcl-2 axis, which may shed new light on the exploration of therapeutic targets for the clinical treatment of GC.

Keywords: autophagy; circRNA; gastric cancer; hsa_circ_0009109; miR-544a-3p.

Figure 1.

Methodological evaluation of the characteristics of hsa_circ_0009109 in GC. (A) Schematic diagram of hsa_circ_0009109 formation. Arrows represent convergent primers and divergent primers designed for hsa_circ_0009109 amplification. (B) PCR products detected using agarose gel electrophoresis analysis. A single band with the length of 131 bp is shown. (C) Verification of the cyclization site of hsa_circ_0009109 using Sanger sequencing. (D) Verification of the loop structure of hsa_circ_0009109 with convergent primers and divergent primers. (E) RNA exonuclease digestion assay. The data are presented as the mean ± SD. ***P < 0.001. GC = gastric cancer, SD = standard deviation.

Figure 2.

Biological role of hsa_circ_0009109 in GC cells. (A) The expression of hsa_circ_0009109 in 80 pairs of GC tissues and matched non-cancerous tissues. (B) qPCR assay demonstrated downregulation of hsa_circ_0009109 expression in five GC cells relative to GES-1 cells. (C) Verification of transfection efficiency using qPCR. (D) and (E) Detection of the cell proliferation ability by CCK-8 and colony formation assays. (F) Measurement of the cell migration and invasion abilities using the transwell assay. Testing of the cell apoptosis rate (G) and cell cycle progression (H) using flow cytometry. (I) The expression of cell cycle- and apoptosis-associated proteins. GAPDH is used as the internal reference of CLEAVED CASPASE 3 and 8, and β-ACTIN is used for the remaining proteins. The data are presented as the mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001. UR = upper right, LR = lower right, OD = optical density, GC = gastric cancer, SD = standard deviation.

Figure 3.

Overexpression of hsa_circ_0009109 blocks tumor growth in the xenograft model. (A) Representative images of subcutaneous xenografts in nude mice in the hsa_circ_0009109 overexpression group (LV-pLCDH-ciR) and empty vector group (LV-NC). (B) The expression of hsa_circ_0009109 in resected tumors of nude mice. (C) A growth curve of the tumor growth in nude mice. The volume of subcutaneous xenografts was recorded every week. (D) The tumor was weighed after subcutaneous xenografts resection. (E) H&E staining of the resected tumors. (F)–(H) Expression levels of Ki67, CLEAVED CASPASE 3, and CLEAVED CASPASE 8 using immunohistochemistry. The data are presented as the mean ± SD. **P < 0.01; ***P < 0.001. H&E = hematoxylin and eosin, SD = standard deviation.

Figure 4.

MiR-544a-3p is sponged by hsa_circ_0009109 in GC. (A) Detection of hsa_circ_0009109 location in SGC-7901 and MKN-45 cells using nucleoplasm separation assay. (B) Localization of hsa_circ_0009109 using FISH in SGC-7901 cells. (C) The expression of miR-544a-3p in five GC cells. (D) Expression of miR-544a-3p in hsa_circ_0009109-overexpressed BGC-823 cells and hsa_circ_0009109-silenced SGC-7901 cells. (E) The expression of miR-544a-3p in 58 pairs of GC tissues. (F) A negative correlation between hsa_circ_0009109 and miR-544a-3p was observed by using Spearman correlation analysis. (G) Putative binding sites of miR-544a-3p to hsa_circ_0009109. The luciferase assay was adopted to verify the bind sites between hsa_circ_0009109 and miR-544a-3 in HEK-293T cells. The data are presented as the mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001. WT = wild-type, MUT = mutant, FISH = fluorescence in situ hybridization, GC = gastric cancer, SD = standard deviation.

Figure 5.

Hsa_circ_0009109 suppresses the malignant phenotype of GC cells via sponging miR-544a-3p. (A) and (B) CCK-8 and colony formation assays were used to evaluate the proliferation ability of BGC-823 cells co-treated with miR-544a-3p mimic (miR-mimic) and overexpression vector (pLCDH-ciR) of hsa_circ_0009109. (C) and (D) Flow cytometry analysis was performed to detect the ratio of cell apoptosis and proportion of cells in the S-phase in dual-transfected BGC-823 cells. All data are presented as the mean ± SD, *P < 0.05; **P < 0.01. UR = upper right, LR = lower right, GC = gastric cancer, SD = standard deviation.

Figure 6.

Hsa_circ_0009109 regulates tumor progression through inhibiting autophagy via the miR-544a-3p/bcl-2 axis. (A) Putative binding sites of miR-544a-3p to BCL-2 using TargetScan. The luciferase assay was performed to verify the bind sites between miR-544a-3 and 3'-UTR of BCL-2 in HEK-293 T cells. (B) and (C) The levels of expression of BCL-2, Beclin1, ATG3, ATG5, ATG7, ATG10, and ATG12 were detected using qRT–PCR in hsa_circ_0009109-overexpressed BGC-823 cells and hsa_circ_0009109-silenced SGC-7901 cells. (D) Immunocytochemical analysis of autophagosomes in BGC-823 cells transfected with overexpression vector of hsa_circ_0009109 (pLCDH-ciR) or treated with 3-MA (2 mM). (E) Expression of autophagy-associated proteins in BGC-823 cells co-treated with pLCDH-ciR and 3-MA (2 mM). (F) Autophagosomes viewed using electron microscopy in hsa_circ_0009109-silenced SGC-7901 cells. The arrows point to the typical autophagosomes. (G) Immunocytochemical analysis of autophagosomes and lysosomes. Anti-LC3 and anti-LAMP1 antibodies were used to mark autophagosomes (LC3B, red fluorescent labeling) and lysosomes (LAMP1, green fluorescent labeling), respectively. (H) Autophagosomes in BGC-823 cells and SGC-7901 cells were observed using an electron microscope. (I) Autophagy-related proteins detected using Western blotting. All data are presented as the mean ± SD. *P < 0.05; **P < 0.01. WT = wild-type, MUT = mutant, SD = standard deviation.