MicroRNA-302 replacement therapy sensitizes breast cancer cells to ionizing radiation

Abstract

Purpose: Solid tumors can be resistant or develop resistance to radiotherapy. The purpose of this study is to explore whether microRNA-302 is involved in radioresistance and can be exploited as a sensitizer to enhance sensitivity of breast cancer cells to radiation therapy.

Methods: MiR-302 expression levels in radioresistant cell lines were analyzed in comparison with their parent cell lines. Furthermore, we investigated whether enforced expression of miR-302 sensitized radioresistant breast cancer cells to ionizing radiation in vitro and in vivo.

Results: MiR-302 was downregulated in irradiated breast cancer cells. Additionally, the expression levels of miR-302a were inversely correlated with those of AKT1 and RAD52, two critical regulators of radioresistance. More promisingly, miR-302a sensitized radioresistant breast cancer cells to radiation therapy in vitro and in vivo and reduced the expression of AKT1 and RAD52.

Conclusion: Our findings demonstrated that decreased expression of miR-302 confers radioresistance and restoration of miR-302 baseline expression sensitizes breast cancer cells to radiotherapy. These data suggest that miR-302 is a potential sensitizer to radiotherapy.

Fig. 1. Expression levels of miR-302a are inversely correlated with the levels of AKT1 and RAD52 in radioresistant breast cancer cell lines

(a) Expression levels of miR-302a determined by RT-PCR are decreased in the radioresistant breast cancer cell lines MDA-MB-231RR and SKBR3RR. Conversely, mRNA expression levels of AKT1 and RAD52 were upregulated in radioresistant cell lines compared to their parental cells. β-actin was used as a loading control. (b) Western blot analysis shows that p-AKT1, total AKT1, and RAD52 proteins are elevated in radioresistant breast cancer cells compared to their counterparts. β-actin was used as a loading control.

Fig. 2. Predicted target sites of miR-302 in 3’ UTRs of AKT1 and RAD52 genes

(a) The putative targeted sites in the 3’ UTRs of AKT1 and RAD52. (b) and (c) Luciferase reporter assay results. MDA-MB-231 cells were transfected with the firefly luciferase reporter plasmid containing partial 3’-UTR of AKT1 (b) or RAD52 (c) with (3’UTR) or without (Mut 3’UTR) the putative miR-302 binding site. Blank vector was used as mock control (Mock). Luciferase activity was measured at 48 h post the co-transfection of luciferase reporter vector with miR-302a mimics. *P<0.01 compared to Mut UTR.

Fig. 3. Overexpression of miR-302 reduced the expression of AKT1 and RAD52

(a) Pre-miRNA double-strand oligo sequence inserted into a miRNA expression plasmid, Block-iT Pol II miR RNAi Expression Vector (Invitrogen). (b) Representative image (GFP) shows the transfection efficiency of the vectors after selection. (c) Levels of miR-302a were increased in miR-302a plasmid-transfected MDA-MB-231RR cells compared to mock MDA-MB-231RR cells. Levels of miR-302a were not significantly increased in control vector-trasfected MDA-MB-231 RR cells compared to mock MDA-MB-231RR cells (d) Levels of phosphorylated AKT1, total AKT1 and RAD52 proteins were reduced in miR-302a plasmid-transfected MDA-MB-231RR cells compared to control vector-transfected or mock MDA-MB-231RR cells. β-actin was used as a loading control.

Fig. 4. MiR-302 sensitized radioresistant tumor cells to irradiation in vitro

(a) miR-302a sensitized MDA-MB-231RR cells to irradiation in vitro. MiR-302a vectors or control vectors were transfected into MDA-MB-231RR cells. Cells were irradiated with an increasing dose of X-ray. A clonogenic assay was performed. Survival fractions shown are the mean and SE from three independent experiments. (b) Similarly, the overexpression of miR-302a increased radiosensitivity of SKBR3RR cells compared to their counterparts in vitro.

Fig. 5. Effects of miR-302 overexpression to radiosensitivity in vivo

(a) Mean xenograft tumor volume of each group at each time point. The day, when mice were irradiated, was designated as Day 0. Bars mean standard errors. Both hind legs of mice in two groups were injected with MDA-MB-231RR cells with or without the transfection of miR-302a vectors. The left hind leg that bore the developed tumor was irradiated at 5 Gy and the right hind legs were used as the mock-irradiated (1Gy) control. The mice were sacrificed at 12 days after irradiation and the tumors were removed for weight comparison. P<0.05. (b) Representatives of xenograft tumors from two groups irradiated with 5 Gy at final time point. (c) Comparison of xenograft tumor weight from two groups irradiated with 5 Gy. *P<0.01. (d) Comparison of mRNA expression levels of miR-302a, AKT-1, and RAD52 in xenografts from two groups irradiated with 5 Gy, *P<0.01.