CircRNA.0007127 triggers apoptosis through the miR-513a-5p/CASP8 axis in K-562 cells

Abstract

BACKGROUND: Circular RNAs (circRNAs) are covalently closed single-stranded RNAs with multiple biological functions. CircRNA.0007127 is derived from the carbon catabolite repression 4-negative on TATA-less (CCR4-NOT) complex subunit 2 (CNOT2), which was found to regulate tumor cell apoptosis through caspase pathway. METHODS: Potential circRNA.0007127 target microRNAs (miRNAs) were analyzed by miRanda, TargetScan, and RNAhybrid software, and the miRNAs with binding sites for apoptosis-related genes were screened. The roles of circRNA.0007127 and its downstream target, microRNA (miR)‍-513a-5p, were validated by quantitative real-time polymerase chain reaction (qPCR), flow cytometry, mitochondrial membrane potential, immunofluorescence, western blot, and caspase-8 (CASP8) protein activity in vitro in H₂O₂-induced K-562 cells. The circRNA.‍0007127‍‒‍miR-513a-5p and CASP8‍‒‍miR-513a-5p interactions were verified by luciferase reporter assays. RESULTS: Silencing circRNA.0007127 decreased cell apoptosis by inhibiting CASP8 pathway activation in K-562 cells. Compared with the control group, the expression of CASP8 was reduced by 50% and the 43-kD fragment of CASP8 protein was significantly reduced (P≤0.05). The luciferase reporting assay showed that circRNA.0007127 combined with miR-513a-5p or CASP8, with extremely significant differences (P≤0.001). The overexpression of miR-513a-5p inhibited the gene expression level of CASP8 in a human myeloid leukemia cell model (75% change) and the level of a 43-kD fragment of CASP8 protein (P≤0.01). The rescue experiment showed that cotransfection with circRNA.0007127 small-interfering RNA (siRNA) and the miR-513a-5p inhibitor increased CASP8 gene expression and the apoptosis rate, suggesting that the miR-513a-5p inhibitor is a circRNA.0007127 siRNA antagonist. CONCLUSIONS: CircRNA.0007127 regulates K-562 cell apoptosis through the miR-513a-5p/CASP8 axis, which can serve as a novel powerful molecular target for K-562 cells.

Keywords: Apoptosis; Caspase-8 (CASP8); CircRNA.0007127; K-562 cells; miR-513a-5p.

Fig. 1. Pathway prediction and identification of circRNA.0007127. (a) Bioinformatics analysis of the potential target miRNAs of circRNA.0007127 by miRanda, TargetScan, and RNAhybrid. (b) Network interaction between circRNA.0007127 and target miRNAs, as well as the downstream mRNAs of these miRNAs. (c) CircRNA.0007127 is formed from the human CNOT2 gene through back-splicing. (d) Schematic diagram of the circularization of exon 2 and exon 3 of CNOT2-forming circRNA.0007127 (the arrowhead shows the junction), where the back-splice junction site (arrow) of circRNA.0007127 was detected by Sanger sequencing. (e) The divergent primers detected circRNA.0007127 in cDNA but not in gDNA. (f) The expression of circRNA.0007127 and CNOT2 mRNAs treated or not treated with RNase R was determined by qPCR. The circRNA.0007127 was resistant to RNase R. Data were shown as mean±standard error of the mean (SEM), n=3. ^*** P<0.001. CircRNA: circular RNA; miRNA: microRNA; mRNA: messenger RNA; CNOT2: carbon catabolite repression 4-negative on TATA-less (CCR4-NOT) complex subunit 2; cDNA: complementary DNA; gDNA: genomic DNA; RNase: ribonuclease; qPCR: quantitative real-time polymerase chain reaction; miR: microRNA; chr: chromosome; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; ns: not significant.

Fig. 2. Suppression of apoptosis in K-562 cells by knockdown of circRNA.0007127. (a) Schematic illustration of siRNA and target sites on circRNA. (b) Expression analyses of three different circRNA-targeting siRNAs in K-562 cells, showing that siRNA-1 had a high knockdown efficiency. (c) The expression of CNOT2 after siRNA transfection was detected by RT-qPCR. (d) Quantification of CASP8, Bax, FADD, CASP3, Bad, and CASP6 in K-562 cells treated with 1.76 mmol/L H₂O₂. (e) Flow cytometry assay showing the exposure of phosphatidylserine on the cell membrane. (f, g) H₂O₂-induced K-562 cells were transfected with siRNA-1, followed by JC-1 fluorescent mitochondrial imaging. The transformation of JC-1 from red fluorescence to green fluorescence was used as an indicator of early apoptosis (scale bar=40 μm). Data were shown as mean±standard error of the mean (SEM), n=3. ^* P<0.05, ^** P<0.01, ^*** P<0.001. CircRNA: circular RNA; siRNA: small-interfering RNA; CNOT2: carbon catabolite repression 4-negative on TATA-less (CCR4-NOT) complex subunit 2; RT-qPCR: reverse transcription-quantitative real-time polymerase chain reaction; CASP8: caspase-8; Bax: Bcl-2-associated X; FADD: Fas-associated protein with death domain; Bad: Bcl-2-associated agonist of cell death; NC: negative control; ns: not significant; 7AAD: 7-amino-actinomycin.

Fig. 3. Effects of circRNA.0007127 on apoptosis by suppressing the expression of CASP8. (a) The mRNA expression levels of CASP8, CASP3, Bad, Bax, CASP6, and FADD after knockdown by circRNA.0007127 were determined by qPCR. (b) Representative western blot images showing the protein expression of CASP8, CASP3, p-FADD, and CASP6 in K-562 cells treated with H₂O₂ and siRNA NC or siRNA-1 for 48 h. GAPDH was used as a loading control. (c) The histogram analysis of the western blot in (b) was performed. (d) Immunofluorescence photomicrographs of CASP8 and CASP3 in circRNA.0007127 siRNA-1 (or siRNA NC)‍-transfected K-562 cells followed by treatment with H₂O₂. Red, green, and blue represent CASP8, CASP3, and nuclei, respectively (scale bar=40 μm). (e) The integrated optical density of CASP8 or CASP3 in (d) was quantified. (f) The hydrolytic activity of CASP8 in K-562 cells with different treatments was measured by absorption value analysis. Data were shown as mean±standard error of the mean (SEM), n=3. ^* P<0.05, ^** P<0.01, ^*** P<0.001. CircRNA: circular RNA; qPCR: quantitative real-time polymerase chain reaction; CASP8: caspase-8; Bax: Bcl-2-associated X; FADD: Fas-associated protein with death domain; Bad: Bcl-2-associated agonist of cell death; siRNA: small-interfering RNA; NC: negative control; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; p-FADD: phospho-FADD; ns: not significant; DAPI: 4,6-diamidinos-2-phenylindole; pNA: p-nitroaniline.

Fig. 4. CircRNA.0007127-targeted hsa-miR-513a-5p. (a) The seed region of miR-513a-5p, the binding sites of circRNA.0007127 with miR-513a-5p, and those of CASP8 with miR-513a-5p. (b) Schematic diagram of the hsa-miR-513a-5p binding sequences of C7127-WT-psiCHECK2.0 and C7127-MUT-psiCHECK2.0. Here, C7127-WT-psiCHECK2.0 represents the WT vector, and C7127-MUT-psiCHECK2.0 represents the MUT vector. (c) MiR-513a-5p mimic or NC as well as miR-513a-5p inhibitor or NC was cotransfected into HEK-293T cells with luciferase reporter containing C7127-WT-psiCHECK2.0 or C7127-MUT-psiCHECK2.0. The luciferase activity of each group was analyzed. Here, psiCHECK2.0 represents the blank vector. Data were shown as mean±standard error of the mean (SEM), n=3. ^** P<0.01. CircRNA: circular RNA; miRNA: microRNA; Mimic NC: miRNA mimic negative control; Inhibitor NC: miRNA inhibitor negative control; CASP8: caspase-8; WT: wild type; MUT: mutant; NC: negative control; UTR: untranslated region; R/F: relative activity of Renilla luciferase/firefly luciferase.

Fig. 5. Effects of miR-513a-5p on apoptosis by binding to the 3' UTR region of CASP8. (a) Luciferase reporter assay was performed after HEK-293T cells were cotransfected with CASP8-WT-psiCHECK2.0 or CASP8-MUT-psiCHECK2.0 and miR-513a-5p mimic or mimic NC. (b) The effects of miR-513a-5p on CASP8 expression in H₂O₂-induced K-562 cells transfected with mimic or inhibitor were analyzed by qPCR. (c) The absorbance values of K-562 cells in different treatment groups were detected by enzyme plate analyzer, indicating the hydrolytic activity of CASP8. (d) Flow cytometry experiments indicated that the miR-513a-5p mimic reduced the apoptosis of K-562 cells, while its inhibitor further promoted apoptosis. (e) The protein levels of CASP8 and CASP3 were determined by western blotting in apoptotic K-562 cells with knockdown or overexpression of miR-513a-5p (top panel). Representative corresponding densitometry analyses of protein expression levels were performed by western blotting (bottom panel). (f, g) CLSM images showing the expression of CASP8 and CASP3. Red, green, and blue represent CASP8, CASP3, and nuclei, respectively (scale bar=40 μm). Data were shown as mean±standard error of the mean (SEM), n=3. ^* P<0.05, ^** P<0.01. R/F: relative activity of Renilla luciferase/firefly luciferase; CASP8: caspase-8; miR: microRNA; WT: wild type; MUT: mutant; qPCR: quantitative real-time polymerase chain reaction; CLSM: confocal laser scanning microscopy; ns: not significant; 7AAD: 7-amino-actinomycin; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; DAPI: 4,6-diamidinos-2-phenylindole; Mimic NC: miRNA mimic negative control; Inhibitor NC: miRNA inhibitor negative control; UTR: untranslated region; NC: negative control.

Fig. 6. Effects of circRNA.0007127 on apoptosis in H₂O₂-treated K-562 cells via the miR-513a-5p/CASP8 axis. (a) The cell apoptosis in each treatment group was measured by flow cytometry. (b) RT-qPCR showed that the miR-513a-5p inhibitor antagonized the effects of siRNA-1 on CASP8 and CASP3 in apoptotic K-562 cells. (c) siRNA NC, siRNA-1, or siRNA-1 together with miR-513a-5p inhibitor was transfected into apoptotic K-562 cells. Then, the absorbance of each treatment group was detected. (d) Western blotting was performed to measure the protein expression of CASP8 in H₂O₂-induced K-562 cells transfected with siRNA NC, siRNA-1, or siRNA-1 with the miR-513a-5p inhibitor (left panel). Representative corresponding densitometry analyses of protein expressions were performed by western blotting (right panel). (e) CLSM images of K-562 cells labeled with CASP8 or CASP3 in each group. The proportion of CASP3-positive cells increased after transfection with the miR-513a-5p inhibitor. Red, green, and blue represent CASP8, CASP3, and nuclei, respectively (scale bar=40 μm). (f) The integrated optical density of CASP8 or CASP3 in (e) was quantified. (g) The hypothesis diagram illustrates the function and mechanism of circRNA.0007127 in the miR-513a-5p/CASP8 axis apoptosis pathway. Data were shown as mean±standard error of the mean (SEM), n=3. ^* P<0.05, ^** P<0.01, ^*** P<0.001. CircRNA: circular RNA; siRNA: small-interfering RNA; miR: microRNA; CASP8: caspase-8; RT-qPCR: reverse transcription-quantitative real-time polymerase chain reaction; NC: negative control; CLSM: confocal laser scanning microscopy; 7AAD: 7-amino-actinomycin; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; ns: not significant; mRNA: messenger RNA; DISC: death-inducing signaling complex; pNA: p-nitroaniline.