High Expression of circ_0001821 Promoted Colorectal Cancer Progression Through miR-600/ISOC1 Axis

Abstract

It has been reported that circRNAs play an important regulatory role in the progression of colorectal cancer (CRC). However, the molecular role of circ_0001821 in CRC development is unclear. In this study, we aimed to investigate the regulatory role and molecular mechanisms of circ_0001821 in CRC. Reverse transcription-quantitative PCR and western blot assays were used to detect the expression of circ_0001821, miR-600 and isochorismatase domain containing 1 (ISOC1) in CRC tissues as well as its cell lines. Colony formation assay and EDU assay were used to detect the proliferative capacity of cells. Transwell assay was used to assess cell migration and invasion ability. Flow cytometry was used to analyze cell apoptosis. ELISA was used to measure the glycolytic capacity of cells. Dual-luciferase reporter assay and RNA pull-down assay were used to analyze the relationships between circ_0001821, miR-600 and ISOC1. Animal experimentation was used to validate the functional study of circ_0001821 in vivo. Immunohistochemistry (IHC) of Ki67 staining analysis was conducted to assess tumor growth. Circ_0001821 and ISOC1 were significantly increased in CRC tissues and its cell lines, and miR-600 was significantly decreased in CRC tissues and its cell lines. Silencing circ_0001821 inhibited cell proliferation, migration, invasion and glycolytic capacity, while inducing apoptosis. And it could inhibit tumor growth in vivo. Circ_0001821 could act as a sponge for miR-600 to regulate CRC processes. ISOC1 was identified as a downstream regulator of miR-600, also miR-600 could regulate the expression of ISOC1. In addition, circ_0001821 could regulate ISOC1 expression changes through miR-600. Mechanistically, either miR-600 inhibitor or overexpression of ISOC1 could reverse the effects of knockdown of circ_0001821 on cell biological properties. Circ_0001821 regulated the developmental process of CRC through miR-600/ISOC1 axis.

Keywords: Colorectal cancer; ISOC1; circ_0001821; miR-600.

Fig. 1

Circ_0001821 was increased in CRC tissues. A The chromosomal location of circ_0001821 on its host gene TCONS_00015354 was shown, along with the exons contained in circ_0001821. B and C The expression of circ_0001821 was tested in CRC tissues, CRC cell lines (SW620 and HCT116), and matched normal controls by RT-qPCR. ***p < 0.001

Fig. 2

Effect of circ_0001821 silencing on the biological properties of CRC cell lines. A The knockdown efficiency of circ_0001821 was detected by RT-qPCR in SW620 and HCT116 cells. B and C The effect of silencing circ_0001821 on cell proliferation was assessed by colony formation assay and EDU assay. D and E The effect of silencing circ_0001821 on the ability of cells to migrate and invade was examined by transwell assay. F The effect of knockdown of circ_0001821 on the rate of apoptosis was measured by flow cytometry. G and H Glucose uptake and lactate production in CRC cells were detected after silencing circ_0001821. I and J The protein expression of PCNA, MMP2, Bax and HK2 was detected by western blot assay. ***p < 0.001

Fig. 3

Identification of miR-600 as a potential miRNA sponge for circ_0001821. A Binding sites and mutant sites between circ_0001821 and miR-600 were shown. B The expression of miR-600 in tumor and normal tissues was assessed by RT-qPCR. C The expression of miR-600 in NCM460, SW620 and HCT116 cells was assessed by RT-qPCR. D–G Interaction between circ_0001821 and miR-600 was detected by dual-luciferase reporter assay and RNA pull-down assay. H Pearson’s correlation analysis showed the correlated expression levels between circ_0001821 and miR-600. ***p < 0.001

Fig. 4

Effect of circ_0001821 silencing on cell biological properties was reversed by miR-600 inhibitor. A The expression of miR-600 was tested by RT-qPCR in CRC cell lines. B and C The proliferative capacity of the cells was assayed by colony formation assay and EDU assay after transfection with si-NC, si-circ_0001821, si-circ_0001821 + anti-miR-NC or si-circ_0001821 + anti-miR-600 in SW620 and HCT116 cells. D and E Cell migration and invasion capabilities in abovementioned cells were measured by transwell assay. F The apoptotic capacity of the abovementioned cells was detected by flow cytometry. G and H Glucose consumption and lactate production in SW620 and HCT116 cells were assessed. I and J The protein expression of PCNA, MMP2, Bax and HK2 was detected in abovementioned cells using western blot. **p < 0.01, ***p < 0.001

Fig. 5

MiR-600 directly bound to ISOC1. A The binding site between miR-600 and ISOC1 mRNA was shown. B–D The mRNA and protein expression of ISOC1 was detected by RT-qPCR and western blot assay. E and F Interaction relationship between miR-600 and mRNA was measured by dual-luciferase reporter assay. G The protein expression of ISOC1 was measured after transfection with miR-NC or anti-miR-600. H and I The protein expression of ISOC1 was measured by western blot assay after transfection with si-NC, si-circ_0001821, si-circ_0001821 + anti-miR-NC or si-circ_0001821 + anti-miR-600. J and K Pearson’s correlation analysis showed the correlated expression levels between miR-600 and ISOC1 and between circ_0001821 and ISOC1 were shown. **p < 0.01, ***p < 0.001

Fig. 6

Effect of circ_0001821 silencing on cell biological properties was counteracted by overexpression of ISOC1. A Overexpression efficiency of ISOC1 was examined by western blot assay. B–J SW620 and HCT116 cells were transfected with si-NC, si-circ_0001821, si-circ_0001821 + pcDNA or si-circ_0001821 + ISOC1. B and C The proliferative capacity of the cells was measured by colony formation assay and EDU assay. D and E The cell migration and invasion capabilities were assessed by transwell assay. F Flow cytometry was used to detect apoptosis of cells. G and H Glucose consumption and lactate production in CRC cell lines were tested. I and J Western blot assay was used to assess the protein expression of PCNA, MMP2, Bax and HK2. **p < 0.01, ***p < 0.001

Fig. 7

Knockdown of circ_0001821 repressed tumor growth in vivo. A and B Tumor volume and tumor weight in sh-NC group and sh-circ_0001821 group were measured. C Ki-67 levels were assessed by IHC. D and E The expression of circ_0001821 and miR-600 was assessed by RT-qPCR. F and G The mRNA and protein expression of ISOC1 was tested by RT-qPCR and western blot assay. **p < 0.01, ***p < 0.001