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. 2022 Nov;36(11):e24710.
doi: 10.1002/jcla.24710. Epub 2022 Sep 30.

Hsa_hsa_circ_0081069 promotes the progression of colorectal cancer through sponging miR-665 and regulating E2F3 expression

Jingjing Xie  1 , Dan Jin  1 , Jinyin Xu  1 , Fei Yang  1 , Jianying Jin  1
Affiliations

Hsa_hsa_circ_0081069 promotes the progression of colorectal cancer through sponging miR-665 and regulating E2F3 expression

Jingjing Xie et al. J Clin Lab Anal. 2022 Nov.

Abstract

Background: Circular RNAs (circRNAs) have been implicated in the initiation and development of various cancers. This study explored the potential contribution of hsa_hsa_circ_0081069 in the progression of colorectal cancer (CRC).

Methods: The gene expression was analyzed by qRT-PCR. Functional roles of hsa_circ_0081069 were examined by shRNA-mediated silencing using CCK-8 proliferation assay, Transwell migration and invasion assay, tube formation assay. The tumorigenesis and metastasis of CRC cells were assess in a xenograft mouse model.

Results: Hsa_circ_0081069 was significantly upregulated in CRC tissues and cells. Hsa_circ_0081069 knockdown suppressed the proliferation, migration and invasion in CRC cells, as well as the angiogenesis. Silencing hsa_circ_0081069 also impaired the tumorigenesis of CRC cells in a xenograft mouse model. Furthermore, miR-665 was identified as an interacting partner of hsa_circ_0081069, which was negatively regulated by hsa_circ_0081069. miR-665 targeted the mRNA of E2F3 to suppress its expression. We further demonsatred that miR-665/E2F3 axis mediated the functional role of hsa_circ_0081069 in regulating the malignant phenotype of CRC cells.

Conclusions: Collectively, our study suggests that hsa_circ_0081069 could serve as a prognostic marker in progression of CRC. Targeting hsa_circ_0081069 and miR-665/E2F3 axis could serve as potential therapeutic strategies for CRC treatment.

Keywords: E2F3; Hsa_circ_0081069; MiR-665; colorectal cancer; progression.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

FIGURE 1
FIGURE 1
Hsa_circ_0081069 was upregulated in CRC tissues and cells. (A) Schematic illustration of Hsa_circ_0081069 structure. (B) qRT‐PCR analysis of hsa_circ_0081069 expression in CRC cell lines and FHC cells. (C) Cells were treated with Actinomycin D, and the level of Hsa_circ_0081069 and COL1A2 mRNA at different time points were determined by qRT‐PCR. (D) Expression level of hsa_circ_0081069 and COL1A2 mRNA after RNase R treatment was determined by qRT‐PCR. (E) Hsa_circ_0081069 expression was determined by qRT‐PCR in CRC tissues and para‐cancerous tissues. (F) KM‐plotter analysis of the overall survival of CRC patients in Hsa_circ_0081069 high and low expression group. ***p < 0.001
FIGURE 2
FIGURE 2
Knockdown of hsa_circ_0081069 suppressed the proliferation, migration, invasion and angiogenesis. (A) qRT‐PCR analysis of the knockdown efficiency of sh‐hsa_circ_0081069 #1, #2 and #3 in HCT116 and LoVo cells. (B) COL1A2 mRNA expression in sh‐NC and sh‐hsa_circ_0081069 was measured by qRT‐PCR. (C) CCK8 proliferation assay was performed upon hsa_circ_0081069 silencing. (D) EdU incorporation assay was performed upon hsa_circ_0081069 silencing. (E, F) Transwell migration and invasion assays were performed upon hsa_circ_0081069 silencing. (G) Tube formation assay in cells upon hsa_circ_0081069 silencing. *p < 0.05. **p < 0.01. ***p < 0.001
FIGURE 3
FIGURE 3
Hsa_circ_0081069 targeted miR‐665. (A) qRT‐PCR was used to quantify the relative abundance of hsa_circ_0081069 in the nucleus and cytoplasm. (B) The potential interacting miRNAs of hsa_circ_0081069 were predicted via Starbase, circBank, and circIntreactome. (C) RNA pull‐down analysis using biotinylated control oligo or hsa_circ_0081069 probe. (D) RIP assay using IgG or anti‐Ago2 antibody. (E) MiR‐665 expression was assessed in different cell lines by qRT‐PCR. (F) MiR‐665 expression was assessed in CRC tissue and para‐cancerous tissues by qRT‐PCR. (G) Pearson correlation analysis of the correlation between hsa_circ_0081069 and miR‐665 in CRC tissues. (H) Dual luciferase reporter assay using WT or MUT reporter, in the presence of miR‐665 mimic or miR‐NC. (I) miR‐665 expression level was assess by qRT‐PCR upon hsa_circ_0081069 knockdown. ***p < 0.001
FIGURE 4
FIGURE 4
MiR‐665 targeted E2F3. (A) Dual luciferase reporter assay using WT or MUT reporter, in the presence of miR‐665 mimic or miR‐NC. (B) E2F3 expression was determined via qRT‐PCR in CRC tissues and para‐cancerous tissues. (C‐D) Pearson correlation analyses of the correlations between E2F3 and hsa_circ_0081069, or between E2F3 and miR‐665. (E) E2F3 expression determined in different cell lines by qRT‐PCR. (F) E2F3 protein protein level was determined by Western blot upon the transfection of miR‐665 mimic. **/***p < 0.01/0.001
FIGURE 5
FIGURE 5
Hsa_circ_0081069 regulated the malignancy of CRC cells via miR‐665/E2F3 axis. (A) miR‐665 expression level was determined by qRT‐PCR upon miR‐665 inhibitor transfection. (B) E2F3 expression level was determined by Western blot upon the transfection of pcDNA3.1‐E2F3. (C) Western blot analysis of E2F3 expression in the following groups: control, sh‐hsa_circ_0081069, sh‐hsa_circ_0081069 + miR‐665 inhibitor, sh‐hsa_circ_0081069 + pcDNA‐E2F3. (D–H) Cell proliferation assay, EdU incorporation assay, transwell migration and invasion assay, and tube formation assay were performed in cells of different groups: control, sh‐hsa_circ_0081069, sh‐hsa_circ_0081069 + miR‐665 inhibitor, sh‐hsa_circ_0081069 + pcDNA‐E2F3 transfection. ***p < 0.001 compared with sh‐NC, &&/&&& p < 0.01/0.001 compared with sh‐hsa_circ_0081069
FIGURE 6
FIGURE 6
Hsa_circ_0081069 silencing suppressed tumorigenesis in vivo. 12 Male BALB/c mice randomly divided into sh‐hsa_circ_0081069 group (injected with HCT116 cells stably expressing sh‐hsa_circ_0081069), and sh‐NC group (injected with HCT116 cells stably expressing sh‐NC). (A) Tumor volume was measured every 7 days. (B) Tumor weight was determined at day 35. (C) IHC staining of Ki‐67 and E2F3 expression level in the xenograft tissue sections. (D) Hsa_circ_0081069, E2F3 and miR‐665 expression levels were determined by qRT‐PCR. (E) H&E staining of the lung tissue section to evaluate the metastases. **/***p < 0.01/0.001
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