circ86591's suppression of colorectal cancer cell proliferation via cell cycle and metabolic regulation

Wanru Ma; Junhua Hu; Wanru Ma; Junhua Hu

doi:10.48130/epi-0026-0001

Figure 1.

The identification and characteristics of circ86591. (a) Circ86591 was down-regulated in CRC cells with P14AS OE group compared with control group by qRT-PCR. (b) Schematic diagram indicating the circularization of exon8 of CDKN2B-AS formed circ86591. Sanger sequencing demonstrated the presence of circ86591 and its circularization characteristics. (c) SW480 cells were treated with Actinomycin D (5 μg/mL) for duration as indicated, followed by qRT-PCR analysis to examine the expression of circ86591 or GAPDH. (d) Total RNAs extracted from cells were incubated with or without RNase R at 37 °C for duration as indicated, followed by RT-PCR analysis to examine the expression of circ86591 or linANRIL. (e) Circ86591 expression in cytoplasm (GAPDH used as cytoplasmic control) and nucleus (U6 used as nuclear control) of HCT116 and HEK293T cells was detected by qRT-PCR which indicating the circ86591 was enriched in the nuclear of cells. (f) The analysis of the distribution of circ86591 by RNA-FISH assay in HEK293T cells. (g) Biotin-labeled circ86591 pull-down complexes from cell lysate, following mass spectrometry. (h) Western blot analyses of circ86591-EBP1 and ACC1 complexes. (i) High levels of circ86591 were detected by RIP-PCR assay with an anti-EBP1 or anti-ACC1 antibody in HEK293T cells. IgG antibody acted as a negative control.

Figure 2.

Circ86591 affected cell cycle pathway through binding EBP1 protein. (a) The LOVO cells transfected with ASO-NC or ASO-circ86591 were subjected to qRT-PCR to examine the expression of circ86591. The volcano plot showing the expression profile of LOVO cells with ASO-NC and ASO-circ86591. (b) The KOG analysis for genes positively regulated by circ86591 knockdown was shown. (c) The qRT-PCR, and (d) WB assays confirmed the downregulation of CCNE2 and CCNA2 involved in cell cycle in circ86591 knockdown cells. (e) The WB assay was used to detect the protein levels of CCNE2 and CCNA2 after MG132 drug treatment in HCT116 and SW480 cells transfected with ASO-NC or ASO-circ86591. (f) The CCK8 assay exhibited changed in the growth of LOVO and SW480 cells with circ86591 knockdown compared with controls. (g) Flow cytometry indicated that the G₀-G₁ and S phase arrest in the cells with circ86591 knockdown compared with controls.

Figure 3.

circ86591 knockdown influence energy metabolism including nucleotide metabolism in CRC cells. The (a) qRT-PCR, and (b) WB assays confirmed the downregulation of genes involved in nucleotide metabolism in circ86591 knockdown cells. (c) The ATP assay exhibited changes in the ATP content of HCT116 and SW480 cells with circ86591 knockdown compared with controls. (d) The NAD⁺/NADH assay exhibited changes in the content of HCT116 and SW480 cells with circ86591 knockdown compared with controls. (e) The JC-1 MitoMP detection kit showed changes in the mitochondrial membrane potential abilities of HCT116 and SW480 cells with circ86591 knockdown compared with controls. (f) The L-Lactic Acid (LA) colorimetric assay kit exhibited changes in the lactate content of SW480 cells with circ86591 knockdown compared with controls. (g) The mass spectrometry analysis showed that the content of DHAP decreased in the ASO-circ86591 group compared with controls. (h) The genes regulated by circ86591 were up-regulated in COAD tissues compared with normal tissues. (i) The GEPIA database analysis of BPNT1 and SLC25A24 expression were positively correlated with CDKN2B-AS1.

Figure 4.

The effect of circ86591 in vivo. (a) The up-regulated circ86591 in HCT116 and LOVO cells were detected by qRT-PCR and the CCK8 assay exhibiting changes in the growth of CRC cells with circ86591 overexpression compared with controls. (b) The green protein expression of HCT116 and SW480 cells infected with ADV ctrl or ADV circ86591 were showed by fluorescence microscopy. (c) The qRT-PCR assay confirmed the overexpression of circ86591 in ADV ctrl and ADV circ86591 group. (d) NVSG mice were subcutaneously implanted with HCT116 cells, and treated with ADV ctrl or ADV circ86591 for three weeks (once per week) and the tumors were collected and photographed. (e) The volume and weight of tumors as described in (d) was shown. (f) The RNA-seq analysis of P53 gene expression level between ADV ctrl and ADV circ86591 group. (g) The WB assay confirmed the elevation of P53 protein levels in HCT116 cells after infection with ADV circ86591. (h) The P53 and PCNA protein levels in tumor of (d) were detected by the WB assay.

Figure 5.

circ86591 knockdown could increase drug resistance in CRC cells. (a) The CCK8 assay was performed for cell survival after drug treatment in ASO-NC and ASO- circ86591 groups. (b) The CCK8 assay was performed for cell survival after GEM treatment in ADV ctrl and ADV circ86591 groups.

Figure 6.

circ86591 was related to lipid metabolic pathway via analysis of RNA-seq. (a) The KEGG analysis for genes positively regulated by circ86591 knockdown was shown. (b) Fluorescence microscopy was used to observe lipid droplet synthesis in the ASO-circ86591 or ASO-NC group, and three fields of view were randomly selected for fluorescence intensity analysis. (c)The KEGG analysis for genes positively regulated by circ86591 overexpression was shown. (d) Fluorescence microscopy was used to observe lipid droplet synthesis in the ADV circ86591 or ADV ctrl group, and three fields of view were randomly selected for fluorescence intensity analysis. (e) The HCT116 and SW480 cells infected with ADV ctrl or ADV circ86591 combined with DMSO or CMS121 were subjected to cell proliferation assay (up) and WB assay (below). (f) Schematic representation of the functional role of circ86591.