The circular RNA circ-ERBIN promotes growth and metastasis of colorectal cancer by miR-125a-5p and miR-138-5p/4EBP-1 mediated cap-independent HIF-1α translation

Abstract

Background: Circular RNA (circRNAs) and hypoxia have been found to play the key roles in the pathogenesis and progression of cancer including colorectal cancer (CRC). However, the expressions and functions of the specific circRNAs in regulating hypoxia-involved CRC metastasis, and the circRNAs that are relevant to regulate HIF-1α levels in CRC remain elusive.

Methods: qRT-PCR was used to detect the expression of circRNAs and mRNA in CRC cells and tissues. Fluorescence in situ hybridization (FISH) was used to analyze the location of circ-ERBIN. Function-based experiments were performed using circ-ERBIN overexpression and knockdown cell lines in vitro and in vivo, including CCK8, colony formation, EdU assay, transwell, tumor growth and metastasis models. Mechanistically, luciferase reporter assay, western blots and immunohistochemical stainings were performed.

Results: Circ-Erbin was highly expressed in the CRC cells and Circ-Erbin overexpression facilitated the proliferation, migration and metastasis of CRC in vitro and in vivo. Notably, circ-Erbin overexpression significantly promoted angiogenesis by increasing the expression of hypoxia induced factor (HIF-1α) in CRC. Mechanistically, circ-Erbin accelerated a cap-independent protein translation of HIF-1α in CRC cells as the sponges of miR-125a-5p and miR-138-5p, which synergistically targeted eukaryotic translation initiation factor 4E binding protein 1(4EBP-1).

Conclusions: Our findings uncover a key mechanism for circ-Erbin mediated HIF-1α activation by miR-125a-5p-5p/miR-138-5p/4EBP-1 axis and circ-ERBIN is a potential target for CRC treatment.

Keywords: Circular RNA; Colorectal cancer; hypoxia; miRNA sponge.

Fig. 1

Identification of circ-ERBIN as a candidate gene for metastasis of CRC. a Schematic illustration of three circRNAs in ERBIN gene. b RNA expression of the three circRNAs in different CRC cell lines was detected using qRT-PCR. c, d qRT–PCR for the abundance of ERBIN (left) and circ-ERBIN (right) RNA levels in HCT116 and RKO cells treated with RNase R (c) and followed by Sanger sequencing (d). The amount of ERBIN and circ-ERBIN RNA were normalized to the value measured in the mock treatment. e The relative RNA levels of ERBIN and circ-ERBIN were analyzed by qRT-PCR after treatment with Actinomycin D at the indicated time points in HCT116 cells. f, g qRT-PCR (f) and FISH (g) data indicating the circ-ERBIN is abundant in the cytoplasm. f GAPDH and U6 were applied as positive controls in the cytoplasm and nucleus for qRT-PCR experiments, respectively. g 18 s and U6 were applied as positive controls in the cytoplasm and nucleus for Fluorescence in situ hybridization (FISH) experiments, respectively. Nuclei were stained with DAPI. h qRT–PCR for the abundance of circ-ERBIN RNA in the samples of a mouse model of CRC metastasis. i RNA levels of circ-ERBIN in 59 pairs randomly selected CRC samples were detected by the junction primers (P < 0.001). j Expression of circ-ERBIN in different stages of CRC carcinoma samples. Values are the average of three independent experiments. Data are expressed as the mean ± SD, P<0.001

Fig. 2

Overexpression of circ-ERBIN promotes proliferation, migration, invasion of CRC in vitro and in vivo. a-c CCK8, EdU and colony formation assays in circ-ERBIN op stable cells and its control pLCDH cells. d Cell migration and invasion assays were performed and measured in circ-ERBIN op stable cells and its control pLCDH cells. Data are means ± SD. *, P<0.05 vs pLCDH. e-g HCT116 stably expressed circ-ERBIN or pLCDH vector were injected into BALB/C nude mice. Tumor volumes and weights were analyzed. h-i HCT116 cells (2 × 10⁶ cells/mouse) with circ-ERBIN overexpression were injected through tail vein into nude mice to produce lung metastasis model of CRC. h Eight weeks later, mice were sacrificed and lungs were isolated (upper panel). Lung weight/Body weight value (mg/g) was calculated (bottom panel). i Images of H&E staining are shown and tumor area was measured

Fig. 3

Circ-ERBIN knockdown suppresses CRC cells proliferation, migration and invasion in vitro and in vivo. a Transwell migration assays in HCT116 and RKO cells after circ-ERBIN siRNAs transfection. b-d CCK8, EdU and colony formation assays in HCT116 cells after circ-ERBIN knockdown. e HCT116 sh-circ-ERBIN stable cells were analyzed for their in vitro migration and invasion using transwell chambers and invasion chambers, respectively. f-g HCT116 sh-circ-ERBIN stable cells were injected into BALB/C nude mice (n = 6 for each group). Tumor volumes and weights were monitored. h The number of metastases in mice injected with sh-circ-ERBIN #1/#3 stable cells through tail vein into nude mice to produce lung metastasis model of CRC. Images of H&E staining (Left) are shown and tumor area was measured (Right). Data are means ± SD. * and #, P<0.05 vs LV3 or Oligo

Fig.  4

Circ-ERBIN exerts an oncogenic role via alleviating HIF-1α levels in CRC cells. a HE staining and IHC experiments for CD31 and HIF-1α in subcutaneous tumors formed by circ-ERBIN op and pLCDH cells. b Microvascular densities (MVD) of the indicated xenograft tumors were detected by CD31 staining. Scale bars: 100 μm. P<0.001. c Correlation between CD31 positive number and HIF-1α expression. R = 0.198, P = 0.015. d IHC score for HIF-1α in the indicated subcutaneous tumors. P<0.001. e Western blot experiments were performed using subcutaneous tumor tissues formed by circ-ERBIN op or sh-circ-ERBIN cells, respective antibodies were used. f, g Western blot (f) and qRT-PCR (g) experiments were performed using stable overexpression or knockdown of circ-ERBIN cell lines. h, i Circ-ERBIN op (h) or knockdown (i) cells were treated with Cocl₂ for 24 h and followed by CHX treatment for different minutes. Western blot experiments were performed to test the degradation rate of HIF-1α. j, k Circ-ERBIN op (j) or knockdown (k) cells were treated with Cocl2 for 24 h and followed by MG132 treatment for 4 h

Fig. 5

Circ-ERBIN elevates the expression of HIF-1α via 4EBP-1. a Venn diagram for the putative target miRNAs of circ-ERBIN. b Venn diagram for the downstream targets of circ-ERBIN targeted miRNAs. c Schematic diagram of the predicted miR-125a-5p and miR-138-5p binding sites for 4EBP-1. d Circ-ERBIN overexpression (pLCDH/circ-ERBIN) stable cells were used to perform subcutaneous tumor model and tail vein injection metastasis model, respectively. Immunohistochemical staining of 4EBP-1 were presented. Scale bars, 100 μm. e, f 4EBP-1 siRNA or 4EBP-1 plasmids were transfected into HCT116 circ-ERBIN op or knockdown cells, respectively. Western blots were performed to determine the corresponding proteins. g, h Western blot experiments were performed using HCT116 circ-ERBIN stable overexpression or knockdown cell lines, antibodies with different phosphorylation sites of 4EBP-1 were used respectively. i Circ-ERBIN op or pLCDH cells were immunopreipitated (IP) with IgG or eIF4G antibody, and the immunoprecipitates were analyzed by western blot. j Circ-ERBIN op or pLCDH cells were transiently transfected with pRnegR or pRhif-1αF plasmids. Renilla and firefly luciferase activities were determined after 24 h tranfection, and IRES activities represented as ratios of Firefly to Renilla luciferase. Data are means ± SD. *, P<0.05.

Fig. 6

Circ-ERBIN elevates HIF-1 α expression by miR-125a-5p/miR-138-5p/4EBP-1 signaling. a Luciferase activity of LUC-circ-ERBIN WT or LUC-circ-ERBIN Mutant in HCT116 cells after co-transfection with miR-125a-5p or/and miR-138-5p mimics. b qRT-PCR was performed to detect related miRNAs and 4EBP-1 levels using mouse subcutaneous tumor samples. c, d qRT-PCR experiments were performed to detect miR-125a-5p and miR-138-5p mRNA levels using clinical CRC patient samples (n = 32). The correlations between circ-ERBIN and miR-125a-5p or miR-138-5p were represented. All the qRT-PCR experiments were normalized using GAPDH as an endogenous control. Data are means ± SD. P<0.05. e Luciferase activity of 4EBP-1 3’UTR constructs harboring wide type sequence or mutant sequence were analyzed after being transfected with miR-125a-5p or miR-138-5p mimics alone or together. β-gal was transfected as control. f miR-125a-5p and miR-138-5p mimics were transfected into circ-ERBIN stable cell lines alone or together. Western blots were performed to analyze the protein of 4EBP-1 and HIF-1α. g miR-125a-5p or miR-138-5p inhibitor was transfected alone or together into HCT116 sh-circ-ERBIN stable cell lines. Western blots were performed to analyze the protein levels of 4EBP-11 and HIF-1α. h, i The correlations between 4EBP-1 and miR-125a-5p or miR-138-5p were represented. j The correlation between circ-ERBIN and 4EBP-1 was represented. All the qRT-PCR experiments were normalized using GAPDH as an endogenous control. Data are means ± SD. P<0.05.

Fig. 7

Circ-ERBIN accelerates the growth and metastasis of CRC by activating HIF-1α signaling through the circ-ERBIN/miR-125a-5p/miR-138-5p/ 4EBP-1 pathway. a Transwell assays for HCT116 cells that stably overexpressed circ-ERBIN or co-transfected with miR-125a-5p or miR-138-5p mimics after 2 days. b MiR-125a-5p or miR-138-5p inhibitor were transfected alone or together into HCT116 sh-circ-ERBIN #1 stable cell lines for 2 days followed by the analysis of cell migration using transwell assays. Cells were stained by Giemsa’s staining and visualized under a phase-contrast microscope. c-e HCT116 stably expressed circ-ERBIN or pLCDH vector were injected into BALB/C nude mice (n = 5 for each group). AgomiRs were injected into tumors as represented. The tumors were excised and photographed, and the size and weight of tumors were presented. f Representative immunohistochemical stainings of 4EBP-1 and HIF-1α in subcutaneous tumors with agomiRs injection as described. Scale bars, 100 μm. g A schematic model displaying the role of circ-ERBIN/miR-125a-5p/miR-138-5p/ 4EBP-1/HIF-1α axis in CRC metastasis.