Knockdown of miR-27a sensitizes colorectal cancer stem cells to TRAIL by promoting the formation of Apaf-1-caspase-9 complex

Abstract

MicroRNAs have been proved to participate in multiple biological processes in cancers. For developing resistance to cytotoxic drug, cancer cells, especially the cancer stem cells, usually change their microRNA expression profile to survive in hostile environments. In the present study, we found that expression of microRNA-27a was increased in colorectal cancer stem cells. High level of microRNA-27a was indicated to induce the resistance to TNF-related apoptosis-inducing ligand (TRAIL). Knockdown of microRNA-27a resensitized colorectal cancer stem cells to TRAIL-induced cell death. Mechanically, the gene of Apaf-1, which is associated with the mitochondrial apoptosis, was demonstrated to be the target of microRNA-27a in colorectal cancer stem cells. Knockdown of microRNA-27a increased the expression level of Apaf-1, thus enhancing the formation of Apaf-1-caspase-9 complex and subsequently promoting the TRAIL-induced apoptosis in colorectal cancer stem cells. These findings suggested that knockdown of microRNA-27a in colorectal cancer stem cells by the specific antioligonucleotides was potential to reverse the chemoresistance to TRAIL. It may represent a novel therapeutic strategy for treating the colorectal cancer more effectively.

Keywords: Apaf-1; TRAIL; caspase-9; colorectal cancer stem cells; miR-27a.

Figure 1. Overexpresion of miR-27a in colorectal cancer stem cells

(A) Flow cytometry analysis was performed to detect the populations of CSCs and non-CSCs in HT29 and SW48 cell lines. (B) Expression of miR-27a in FHC, HT29 and SW480 CSCs and non-CSCs was measured by using qRT-PCR analysis. *P < 0.05 vs. FHC cells. ^#P < 0.05. (C) Expression of miR-27a in normal tissues, colorectal CSCs and non-CSCs was measured by using qRT-PCR analysis. *P < 0.05 vs. normal tissues. ^#P < 0.05 vs. colorectal non-CSCs.

Figure 2. MiR-27a antioligonucleotides sensitized colorectal CSCs to TRAIL

(A) CCK-8 assays were performed to evaluate the sensitivity of HT29 and SW480 CSCs and non-CSCs to TRAIL. *P < 0.05. (B) After transfection with miR-27a antioligonucleotides, relative expression of miR-27a in HT29 non-CSCs, HT29 CSCs, SW480 non-CSCs and SW480 CSCs was detected by qRT-PCR analysis. *P < 0.05 vs. NCO group. (C) CCK-8 assays were performed to evaluate the effect of miR-27a antioligonucleotides on TRAIL-induced cell death in HT29 CSCs and non-CSCs. *P < 0.05. (D) CCK-8 assays were performed to evaluate the effect of miR-27a antioligonucleotides on TRAIL-induced cell death in SW480 CSCs and non-CSCs. *P < 0.05.

Figure 3. Effect of miR-27a antioligonucleotides and TRAIL on the activation of caspases and apoptosis in colorectal CSCs

(A) HT29 and SW480 CSCs were treated with miR-27a antioligonucleotides and TRAIL (10 mg/ml). Cell apoptosis was measured by flow cytometry analysis. *P < 0.05 vs. NCO group, ^#P < 0.05 vs. TRAIL + NCO group. (B) HT29 and SW480 CSCs were treated with miR-27a antioligonucleotides and TRAIL (10 mg/ml). Western blot analysis was performed to evaluate the activation of caspase-9 and caspase-3. (C) Cleavage of caspase-8 in HT29 and SW480 CSCs was detected by western blot assays. (D) After separation of mitochondria, cytochrome c in mitochondria and cytoplasm was detected by western blot analysis.

Figure 4. MiR-27a antioligonucleotides increase the expression of Apaf-1 in colorectal CSCs

(A) Apaf-1 mRNA 3′ UTR contained conservative sequence at the miR-27a binding site predicted by the TargetScan database. (B) Expression of Apaf-1 in FHC, HT29 and SW480 CSCs and non-CSCs. (C) Colorectal patients’ tumor and adjacent normal tissues were digested by using collagenase type III to obtain the single cell suspension. After sorting, expression of Apaf-1 in normal colorectal tissue cells, colorectal CSCs and non-CSCs was evaluated by western blot analysis. (D) Luciferase reporter assay in HT29 and SW480 CSCs. The wild or mutant type of Apaf-1 3′ UTR was cloned into pMIR-REPORT plasmid and co-transfected with miR-27a mimics or antioligonucleotides. Dual-Luciferase Reporter Assay System was used to detect the luciferase activities. *P < 0.05. ^#P < 0.05. (E) Effect of miR-27a mimics and antioligonucleotides on changing the expression of Apaf-1 in HT29 CSCs and non-CSCs.

Figure 5. MiR-27a antioligonucleotides sensitized colorectal CSCs to TRAIL by increasing the expression of Apaf-1

(A) Effect of miR-27a antioligonucleotides and Apaf-1 siRNA on changing the expression of Apaf-1 was evaluated by western blot analysis. (B) HT29 and SW480 CSCs were treated with NCO, miR-27a antioligonucleotides, Apaf-1 siRNA and TRAIL (10 mg/ml). Cell viability was measured by using CCK-8 assays. *P < 0.05 vs. NCO group, ^#P < 0.05 vs. TRAIL + NCO group, ^&P < 0.05 vs. miR-27a antioligonucleotides + TRAIL group. (C) HT29 and SW480 CSCs were treated with NCO, miR-27a antioligonucleotides, Apaf-1 siRNA and TRAIL (10 mg/ml). Cell apoptosis was measured by flow cytometry. *P < 0.05 vs. NCO group, ^#P < 0.05 vs. TRAIL + NCO group, ^&P < 0.05 vs. miR-27a antioligonucleotides + TRAIL group.

Figure 6. MiR-27a antioligonucleotides promote the formation of Apaf-1-caspase-9 complex in TRAIL-treated colorectal CSCs

(A) After treatment with miR-27a antioligonucleotides, Apaf-1 siRNA and TRAIL, cleavage of caspase-8 was evaluated by western blot analysis in HT29 and SW480 CSCs. (B) Mitochondrial membrane potential (∆ Ψ_m) was detected by using JC-1 on flow cytometry. (C) After separation of mitochondria, cytochrome c in mitochondria and cytoplasm was detected by western blot analysis. (D) Co-immunoprecipitation and western blot analysis was performed to evaluate the formation of Apaf-1-caspase-9 complex. (E) Cleavage of caspase-9 and caspase-3 in HT29 and SW480 CSCs was evaluated by western blot analysis.

Figure 7. Schema of the mechanisms implicated in miR-27a antioligonucleotides-sensitized apoptosis in TRAIL-treated colorectal CSCs

MiR-27a antioligonucleotides promote TRAIL-dependent formation of Apaf-1/caspase-9 complex by increasing the expression of Apaf-1. As a result, caspase-9 is triggered, followed by activation of caspase-3 and occurrence of apoptosis finally.