EIF4A3-induced circ_0084615 contributes to the progression of colorectal cancer via miR-599/ONECUT2 pathway

Abstract

Background: Colorectal cancer (CRC) is a common malignant tumor. Circular RNAs (circRNAs) have been reported to take part in the progression of CRC. However, the functions of circ_0084615 in CRC development are still undefined. In this study, we aimed to explore the functions and underlying mechanisms of circ_0084615 in CRC.

Methods: qRT-PCR, western blot assay and IHC assay were utilized for the levels of circ_0084615, miR-599, ONECUT2 or EIF4A3. 5-ethynyl-2'-deoxyuridine (EdU) assay and colony formation assay were conducted for cell proliferation ability. Wound-healing assay and transwell assay were applied to evaluate cell migration and invasion. Tube formation assay was used to analyze angiogenesis ability. RNA immunoprecipitation (RIP) assay, RNA pull down assay and dual-luciferase reporter assay were used to analyze the relationships of circ_0084615, miR-599, ONECUT2 and EIF4A3. Murine xenograft model assay was employed for the role of circ_0084615 in vivo.

Results: Circ_0084615 was elevated in CRC tissues and was linked to TNM stages, lymph node metastasis, differentiation and overall survival rate. Circ_0084615 knockdown inhibited CRC cell proliferation, migration, invasion and angiogenesis in vitro and hampered tumorigenesis in vivo. Circ_0084615 sponged miR-599 and miR-599 inhibition reversed circ_0084615 knockdown-mediated effects on CRC cell growth, motility and angiogenesis. ONECUT2 was identified as the target gene of miR-599. ONECUT2 overexpression recovered the effects of miR-599 on CRC malignant behaviors. Additionally, EIF4A3 induced circ_0084615 expression.

Conclusions: EIF4A3-induced circ_0084615 played an oncogenic role in CRC development via miR-599/ONECUT2 axis.

Keywords: Colorectal cancer; EIF4A3; ONECUT2; circ_0084615; miR-599.

Fig. 1

High expression of circ_0084615 in CRC tissues. (A-C) Volcano plot of differentially expressed circRNAs in CRC. (D) The differentially expressed circRNAs simultaneously predicted in GSE126094, GSE138589 and GSE142837. (E-G) Heatmap analysis showed the high levels of hsa_circ_0000512, hsa_circ_0000467, hsa_circ_0040809 and hsa_circ_0084615 in CRC tissues. (H) The levels of hsa_circ_0000512, hsa_circ_0000467, hsa_circ_0040809 and hsa_circ_0084615 in CRC tissues (n = 8) and normal tissues (n = 8) were detected by qRT-PCR assay. (I) The expression of circ_0084615 in CRC tissues (n = 54) and normal tissues (n = 54) was determined by qRT-PCR assay. (J) The expression of circ_0084615 in CRC patients at different TNM stages (29 patients at TNM stages I + II and 25 patients at stage III) was detected by qRT-PCR assay. (K) The expression of circ_0084615 in CRC patients with (n = 22) or without (n = 32) lymph node metastasis was detected by qRT-PCR assay. (L) The expression of circ_0084615 in CRC patients with well/moderate (n = 34) and poor differentiation (n = 20) was detected by qRT-PCR assay. (M) The overall survival of CRC patients with high or low circ_0084615 expression was analyzed. *P < 0.05, ***P < 0.001

Fig. 2

The expression of circ_0084615 in CRC cells and the features of circ_0084615. (A) The expression of circ_0084615 in FHC, SW620, DLD1, SW480 and HT-29 cells was detected by qRT-PCR assay. (B) Circ_0084615 was formed by gene ASPH. (C) The existence of CIRC_0084615 was detected by qRT-PCR assay with divergent or convergent primers. (D and E) The existence of circ_0084615 in the reverse transcription products using random hexamer primers and oligo (dT)18 primers. (F and G) The levels of circ_0084615 and ASPH in SW620 and DLD-1 cells treated with Act D were examined with qRT-PCR assay. (H and I) The levels of circ_0084615 and ASPH in SW620 and DLD-1 cells treated with or without RNase R were detected by qRT-PCR assay. (J and K) The expression of circ_0084615 in the cytoplasm and nucleus of SW620 and DLD-1 cells was analyzed with subcellular fraction analysis. **P < 0.01, ***P < 0.001

Fig. 3

Circ_0084615 interference suppressed cell proliferation, migration, invasion and angiogenesis in CRC cells. SW620 and DLD-1 cells were introduced with sh-NC or sh-circ_0084615. (A) The expression of circ_0084615 in SW620 and DLD-1 cells was examined by qRT-PCR assay. (B-F) The proliferation, colony formation, migration, invasion and angiogenesis of SW620 and DLD-1 cells were assessed by EdU assay, colony formation assay, wound-healing assay, transwell assay and tube formation assay, respectively. (G and H) The protein levels of ZEB2, E-cadherin, Vimentin and VEGFA in SW620 and DLD-1 cells were measured via western blot assay. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 4

Circ_0084615 bound to miR-599 in CRC cells. (A) The targets of circ_0084615 were predicted by starbase and circinteractome. (B-E) The relationship between circ_0084615 and miR-599 was analyzed by RIP and RNA pull-down assays. (F) The complementary sequences between circ_0084615 and miR-599. (G) The expression of miR-599 in SW620 and DLD-1 cells transfected with miR-NC or miR-599 was detected by qRT-PCR assay. (H and I) The interaction between circ_0084615 and miR-599 was examined by dual-luciferase reporter assay. (J and K) The expression of miR-599 in CRC tissues and cells was determined by qRT-PCR assay. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 5

Inhibition of miR-599 rescued the impacts of circ_0084615 knockdown on CRC cell malignant behaviors. (A) The expression of miR-599 in SW620 and DLD-1 cells transfected with anti-NC or anti-miR-599 was detected by qRT-PCR assay. (B-H) SW620 and DLD-1 cells were transfected with sh-NC + anti-NC, sh-circ_0084615 + anti-NC or sh-circ_0084615 + anti-miR-599. (B-F) The proliferation, colony formation, migration, invasion and angiogenesis of SW620 and DLD-1 cells were evaluated via EdU assay, colony formation assay, wound-healing assay, transwell assay and tube formation assay, respectively. (G and H) The protein levels of ZEB2, E-cadherin, Vimentin and VEGFA in SW620 and DLD-1 cells were measured via western blot assay. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 6

MiR-599 directly interacted with ONECUT2. (A) ONECUT2 was predicted to be the target gene of miR-599. (B-E) The combination between miR-599 and ONECUT2 was assessed by RIP and RNA pull down assays. (F) The binding sites between ONECUT2 and miR-599. (G and H) The combination between miR-599 and ONECUT2 was analyzed by dual-luciferase reporter assay. (I and G) The mRNA and protein levels of ONECUT2 in SW620 and DLD-1 cells transfected with miR-NC or miR-599 were measured by qRT-PCR assay or western blot assay. (K and L) The mRNA and protein levels of ONECUT2 in SW620 and DLD-1 cells transfected with sh-NC + anti-NC, sh-circ_0084615 + anti-NC or sh-circ_0084615 + anti-miR-599 were measured by qRT-PCR assay or western blot assay. (M and N) The expression of ONECUT2 in CRC tissues and normal tissues was examined with qRT-PCR and IHC. (O and P) The mRNA and protein levels of ONECUT2 in FHC, SW620, DLD1, SW480 and HT-29 cells were measured by qRT-PCR assay or western blot assay. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 7

MiR-599 overexpression altered CRC cell growth, motility and angiogenesis by binding to ONECUT2. (A) The protein level of ONECUT2 in SW620 and DLD-1 cells transfected with ONECUT2 or vector was measured via western blot assay. (B-H) SW620 and DLD-1 cells were transfected with miR-NC + vector, miR-599 + vector or miR-599 + ONECUT2. (B-F) The proliferation, colony formation, migration, invasion and angiogenesis of SW620 and DLD-1 cells were investigated by EdU assay, colony formation assay, wound healing assay, transwell assay and tube formation assay, respectively. (G and H) The protein levels of ZEB2, E-cadherin, Vimentin and VEGFA in SW620 and DLD-1 cells were measured via western blot assay. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 8

EIF4A3 regulated circ_0084615 expression. (A) The binding sites of EIF4A3 in the flanking sequences of the ASPH1 mRNA transcript were predicted. (B-D) RIP assay demonstrated the binding between ASPH and EIF4A3. (E-H) The levels of EIF4A3 and circ_0084615 in SW620 and DLD-1 cells transfected with sh-NC, sh-EIF4A3, pcDNA or oe-EIF4A3 was measured through western blot assay or qRT-PCR assay. **P < 0.01, ***P < 0.001

Fig. 9

Deficiency of circ_0084615 suppressed tumor formation in vivo. (A-C) Tumor volume and tumor weight were examined. (D) The levels of ONECUT2, ZEB2, E-cadherin, Vimentin and VEGFA in xenograft tumors were examined with IHC assay. ***P < 0.001

Fig. 10

Schematic diagram of EIF4A3-mediated circ_0084615 in regulating CRC tumorigenesis and metastasis