Targeting miR-5088-5p attenuates radioresistance by suppressing Slug

Abstract

Radiotherapy is widely used for cancer treatment, but paradoxically, it has been reported that surviving cancer cells can acquire resistance, leading to recurrence or metastasis. Efforts to reduce radioresistance are required to increase the effectiveness of radiotherapy. miRNAs are advantageous as therapeutic agents because it can simultaneously inhibit the expression of several target mRNAs. Therefore, this study discovered miRNA that regulated radioresistance and elucidated its signaling mechanism. Our previous study confirmed that miR-5088-5p was associated with malignancy and metastasis in breast cancer. As a study to clarify the relationship between radiation and miR-5088-5p identified as onco-miRNA, it was confirmed that radiation induced hypomethylation of the promoter of miR-5088-5p and its expression increased. On the other hand, miR-5088-5p inhibitors were confirmed to reduce radiation-induced epithelial-mesenchymal transition, stemness, and metastasis by reducing Slug. Therefore, this study showed the potential of miR-5088-5p inhibitors as therapeutic agents to suppress radioresistance.

Keywords: Ang, angiopoietin; CSC, cancer stem-like cell; DBC2, deleted in breast cancer 2; DNMT, DNA methyl transferases; EMT, epithelial-mesenchymal transition; H&E, hematoxylin and eosin; IR, ionizing radiation; MSP, methylation-specific PCR; MTT, methylthiazole tetrazolium; Promoter methylation; Radioresistance; Resistance; Slug; VEGF, vascular endothelial growth factor; miR-5088-5p inhibitor; miRNA, microRNA; siRNA, small-interfering RNA.

Fig. 1

IR increases the biogenesis of miR-5088-5p via suppression of its methylation. (A) qRT-PCR analysis of miR-5088-5p expression in plasma of normal (n = 26) and breast cancer patients with (n = 15) and without (n = 21) radiotherapy. (B) Six-week-old mice were subcutaneously injected with H460 and MDA-MB-231 cells (1 × 10⁷ cells/mouse). At 2 weeks after injection, mice were irradiated with 2.5Gy/day for 3days. After 8weeks, mice were sacrificed, tumor tissue isolated and miR-5088-5p expression was confirmed via qRT-PCR (n = 3 out of 5). (C) After irradiation (5Gy) of H460 and MDA-MB-231 cells, levels of the primary, precursor, and mature forms of miR-5088-5p were determined by qRT-PCR and normalized to that of U6. (D) MSP and qMSP assays for methylation of CpG within the miR-5088-5p promoter region in H460 and MDA-MB-231 cells exposed to IR (5Gy). MSP analysis revealed unmethylated (U) and methylated (M) CpG sites and methylation of the Alu element was quantified via qMSP analysis. IVD, in vitro methylated control; DKO, DNMT1 (−/−) DNMT3b (−/−) double knockout (DKO) in HCT116 cells; ddHO, water control without DNA. (E) Confirmation of miR-5088-5p promoter methylation with MSP and qMSP assays using H460 subcutaneous tumor tissues of mice as for B (n = 5). (F) Immunoblot analysis of DNA methylation-related enzymes (DNMT1, DNMT3a, and DNMT3b) after IR (5 Gy) exposure (left) and quantification of bands by Image J program (right). β-actin was used as a loading control. All experiments were performed three times. Data are presented as the mean ± S.D. *P < 0.05, **P < 0.001, and ***P < 0.001 compared to control. Student's t-test.

Fig. 2

The miR-5088-5p inhibitor reduces IR-induced EMT, migratory ability, and invasiveness. (A–C) After IR (5Gy) exposure, H460 and MDA-MB-231 cells were transfected with or without miR-5088-5p inhibitor (anti-miR-5088-5p). (A) Immunoblot analysis of mesenchymal marker proteins (Slug, Zeb1, Snail, Twist, Slug, Vimentin, and N-cadherin) using β-actin as a loading control. (B) Wound healing assay of the migratory ability of cells (scale bar, 100 μm). (C) Matrigel-transwell assay of invasion ability (scale bar, 100 μm). (D) qRT-PCR analysis of invasion-related enzymes (MMP-2 and MMP-9). All assays were performed three times. The data are presented as the mean ± S.D. P values are *P < 0.05, **P < 0.01, and ***P < 0.001 compared to control; ‡P < 0.05 compared to IR group. Student's t-test.

Fig. 3

The miR-5088-5p inhibitor decreases the IR-induced angiogenesis and stemness maintenance. (A) Tube formation assay of angiogenic ability. After transfecting MDA-MB-231 cells with miR-5088-5p inhibitor or negative control (NC), supernatants were obtained with or without IR treatment. After seeding HUVECs (1 × 10⁴ cells/well) in a matrigel-coated 96-well plate, they were cultured for 16 h with each indicated supernatant. (Scale bar, 100 μm). The average number of capillary branches in six random fields was counted and graphed. (B–E) To determine the effect of miR-5088-5p on IR-induced angiogenesis and stemness maintenance, H460 and MDA-MB-231 cells were transfected with miR-5088-5p inhibitor or negative control and treated with/without IR (5Gy). (B) and (C) qRT-PCR analysis of expression of angiogenesis-related factors including VEGF (B) and Ang2 (C). (D) For sphere formation assay to confirm stemness, miR-5088-5p and IR combined cells were seeded in 100 mm culture dishes (1 × 10⁵ cells per dish) and cultured for 12 days. This experiment was performed in triplicate and the average number of colonies was plotted (Scale bar, 250 ㎛). (E) qRT-PCR analysis of expression of cancer stem-like cell markers, such as Oct4, Nanog, Sox2, CD133, and CD44, normalized to that of U6. Data are presented as mean ± S.D. P values are *P < 0.05, **P < 0.01, and ***P < 0.001 compared to control; ‡P < 0.05, ‡ ‡P < 0.01, and ‡ ‡ ‡P < 0.001 compared to IR group. Student's t-test.

Fig. 4

The miR-5088-5p inhibitor reduces IR-induced pulmonary metastasis. (A) For the animal experiment, H460 cells (2 × 10⁶ cells/mouse) transfected with the miR-5088-5p inhibitor were orthotopically injected into tail veins of nude mice (NC, n = 5; anti-miR-5088-5p, n = 4; NC + IR, n = 6; anti-miR-5088-5p + IR, n = 4). On day 14 of injection, mice were treated three times with IR (2.5Gy/day) and ultimately sacrificed at 56 days. (B) Images of pulmonary metastatic tissues were obtained (Scale bar, 500 mm) and stained with H&E (Scale bar, 500 μm). (C) The number of metastatic lung nodules was counted in xenograft mice. (D) qRT-PCR analysis of miR-5088-5p expression in plasma of the indicated groups of mice. (NC, n = 4; anti-miR-5088-5p, n = 3; NC + IR, n = 5; anti-miR-5088-5p + IR, n = 4). qRT-PCR data were quantified with U6. The data are reported as the mean ± S.D. P values are *P < 0.05 compared to control; ‡ ‡P < 0.01 compared to IR group. Student's t-test.

Fig. 5

Slug act as a key factor in suppression of IR-induced malignancy by miR-5088-5p inhibitor. (A) Using the Oncomine public database (www.oncomine.org; lung (Wachi et al., 2005) and breast (Karnoub et al., 2007) cancers), expression of Slug in tissues of lung and breast cancer patients was determined. (B–E) H460 and MDA-MB-231 cells were transfected with siRNA against Slug or negative control (NC) and treated with or without IR (5 Gy). (B) Expression of EMT markers, Zeb1, Snail, Twist, Vimentin, and N-cadherin was confirmed 48 h after transfection by immunoblot analysis. β-actin was used as loading control. (C–E) Migratory ability (C) and invasiveness (D) were confirmed using wound healing (Scale bar, 100 ㎛) and transwell invasion assays (Scale bar, 100 ㎛), respectively, and stemness maintenance was tested using sphere formation assay (E) (Scale bar, 250 ㎛). The data are reported as the mean ± S.D. P values are *P < 0.05, **P < 0.001, and ***P < 0.001 compared to control; ‡P < 0.05 and ‡ ‡P < 0.01 compared to IR group. Student's t-test.

Fig. 6

The miR-5088-5p inhibitor reduces malignant activity by suppressing Slug. (A–F) H460 and MDA-MB-231 cells were transfected with/without a Slug-overexpressing vector in the presence or absence of miR-5088-5p inhibitor and expression of mesenchymal-related proteins (A), migratory (B) and invasive (C) abilities, and angiogenesis-related factors (D) examined by immunoblot analysis, wound healing (Scale bar, 100㎛), transwell invasion (Scale bar, 100㎛), and qRT-PCR assays, respectively. β-actin was used as loading control for immunoblot analysis. (E–F) Measurement of stemness maintenance by the sphere formation assay (Scale bar, 250㎛) (E) and mRNA expression of cancer stem-like cell markers (F). qRT-PCR data was quantified with U6. The data are presented as the mean ± S.D. P values are *P < 0.05, **P < 0.01, and ***P < 0.001 compared to control; ‡P < 0.05, ‡ ‡P < 0.01, and ‡ ‡ ‡P < 0.001 compared to anti-miR-5088-5p group. Student's t-test.

Fig. 7

Schematic diagram showing that the miR-5088-5p inhibitor suppresses IR-induced tumor malignancy through reduction of Slug expression. The miR-5088-5p inhibitor suppresses IR-induced tumor tumorigenicity, metastasis, and resistance to IR and anticancer drug.