Hsa_circ_0001658 accelerates the progression of colorectal cancer through miR-590-5p/METTL3 regulatory axis

Figure 1 The expression and significance of circ_0001658 in colorectal cancer. A: The microarray data set GES172229 available from Gene Expression Omnibus data showed the differences in circular RNAs (circRNAs) expression in colorectal cancer (CRC) tissues and paracancerous tissues, with a volcanic map drawn; B: Heat map displayed the partially up-regulated circRNAs in CRC tissue; C: Circ_0001658 expression in CRC tissues and paracancerous tissues of 42 cases were under the detection of quantitative real-time polymerase chain reaction; D-E: The sensitivity of circ_0001658 to RNase digestion was detected by RNase-R assay. ^bP < 0.01; ^cP < 0.001. ns: No significance; DW: Down; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase.

Figure 2 Effects of circ_0001658 on the multiplication, apoptosis and cell cycle of colorectal cancer cells. A: Circ_0001658 expressions in normal colorectal epithelial cells and colorectal cancer cell lines (HT29, SW480, LoVo, and DLD-1) were under the determination of quantitative real-time polymerase chain reaction (qRT-PCR); B: Negative control (NC) and circ_0001658 overexpressing plasmids were transfected into HT29 cells respectively, and si-NC, si-circ_1 or si-circ-2 were transfected into SW480 cells. The efficiency was then probed by qRT-PCR; C-E: Cell countering kit-8 assay and flow cytometry ensured the analysis on the effect of overexpression or knockdown of circ_0001658 on the proliferation, apoptosis and cell cycle of HT29 and SW480 cells. ^aP < 0.05; ^bP < 0.01; ^cP < 0.001. NC: Negative control; circ-OE: Circ_0001658 plasmid; si-NC: siRNA negative control; OD: Optical density.

Figure 3 Circ_0001658 targets miR-590-5p. A: Subcellular localization of circ_0001658 in HT29 and SW480 cells was under the detection of a nuclear mass separation assay; B: Online database Circular RNA Interactome predicted the binding site of circ_0001658 to miR-590-5p; C-D: Circ-wild-type and circ-mutant type were co-transfected with miR-negative control and miR-590-5p, respectively, into HT29 and SW480 cells, and the activity was probed by a dual luciferase reporter gene assay; E: The effect of circ_0001658 overexpression or knockdown on miR-590-5p expression was probed by quantitative real-time polymerase chain reaction (qRT-PCR); F: The expression of miR-590-5p in normal tissues and colorectal cancer (CRC) tissues was under the assay of qRT-PCR. G: Pearson’s correlation analysis detected the correlations between circ_0001658 and miR-590-5p expression in CRC tissues. ^bP < 0.01; ^cP < 0.001. ns: No significance; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; WT: Wild-type; MUT: Mutant type; miR-NC: miRNA negative control; circ-OE: Circ_0001658 plasmid; si-NC: siRNA negative control.

Figure 4 Circ_0001658 affects the growth, apoptosis and cycle of colorectal cancer cells by adsorption of miR-590-5p. Circ_0001658 was co-transfected with mimics into HT29 cells and si-circ-2 and inhibitors into SW480 cells. A: miR-590-5p expression was probed by quantitative real-time polymerase chain reaction; B-F: The effects of circ_0001658 and miR-590-5p on cell proliferation, apoptosis and cell cycle were detected by cell countering kit-8 assay and flow cytometry. ^aP < 0.05; ^bP < 0.01; ^cP < 0.001. NC: Negative control; circ-OE: Circ_0001658 plasmid; si-NC: siRNA negative control; OD: Optical density.

Figure 5 Circ_0001658 raises METTL3 expression by adsorption of miR-590-5p. A: The downstream targets of miR-590-5p were under the prediction of the bioinformatics websites StarBase and TargetScan; B and C: Kyoto Encyclopedia of Genes and Genomes and Gene Ontology enrichment analysis of the downstream target of miR-590-5P was performed by the Database for Annotation, Visualization and Integrated Discovery database; D: The binding site of miR-590-5P to METTL3 mRNA 3'untranslated region; E: Western blot assay ensured the analysis on the effect of circ_0001658 and miR-590-5p on the expression of MTL 3; F-G: TTL 3-wild-type and TTL 3-mutant type were co-transfected with mimics or miR-negative control into HT29 and SW480 cells and the luciferase activity was measured; H: The expression of METTL3 mRNA in colorectal cancer (CRC) tissues and paracancerous tissues in 42 cases was probed by quantitative real-time polymerase chain reaction; I-J: The correlation of circ_0001658, miR-590-5p and METTL3 mRNA expression in CRC tissues was analyzed by Pearson's correlation analysis. ^cP < 0.001. ns: No significance; BP: Biological process; CC: Cellular component; MF: Molecular function; WT: Wild-type; MUT: Mutant type; miR-NC: miRNA negative control; circ-OE: Circ_0001658 plasmid; si-NC: siRNA negative control.