Loss of miR-200c-3p promotes resistance to radiation therapy via the DNA repair pathway in prostate cancer

Abstract

Radiotherapy represents a major curative treatment for prostate cancer (PCa), but some patients will develop radioresistance (RR) and relapse. The underlying mechanisms remain poorly understood, and miRNAs might be key players in the acquisition and maintenance of RR. Through their encapsulation in small extracellular vesicles (EVs), they can also be relevant biomarkers of radiation response. Using next-generation sequencing, we found that miR-200c-3p was downregulated in PCa RR cells and in their small EVs due to a gain of methylation on its promoter during RR acquisition. We next showed that its exogenous overexpression restores the radiosensitivity of RR cells by delaying DNA repair through the targeting of HP1α. Interestingly, we also observed downregulation of miR-200c-3p expression by DNA methylation in radiation-resistant lung and breast cancer cell lines. In summary, our study demonstrates that the downregulation of miR-200c-3p expression in PCa cells and in their small EVs could help distinguish radioresistant from sensitive tumor cells. This miRNA targets HP1α to delay DNA repair and promote cell death.

Fig. 1. miR-200c-3p downregulation is associated with radioresistance.

A Radiosensitivity of established radiation-resistant and parental cells was evaluated by colony formation assay. RPF: Radiation Protection Factor. Means and SEM are represented. Wilcoxon test, one side: *p < 0.05, **p < 0.01. n = 6 independent experiments. B Volcano plot showing the pairwise comparisons of the differential expression of miRNAs in DU145 and PC3 sensitive and RR cells. The red dots on the left represent miRNAs that were significantly downregulated in the radioresistant cells compared to the parental cells. In green, miRNAs with a log2FC > 1 or <−1 are indicated. In red, miRNAs with an adjusted p value < 0.05. C Log2 Fold Change of miRNAs expressions in DU145 and PC3 cells compared to DU145 RR and PC3 RR cells measured by RT-qPCR (n > 4). D Characterization of the small EVs released by prostate cell lines: Western blot analysis (3 µg of sample was loaded per well. CD63, CD81, and TSG101 proteins are exosomes markers, and calnexin is a cytosolic protein), scanning electron microscopy (SEM) and TRPS technology. In the electron microscopy image, the arrows indicate intact vesicles, while the arrowhead shows amorphous material compatible with protein aggregates. In the TRPS plot, each replicate is represented by a gray level. E Volcano plot of differentially expressed miRNAs in small EVs from DU145 and DU145 RR cells. The red dots on the left represent significantly downregulated miRNAs in small EVs from the DU145 RR cells, and on the right, the upregulated miRNAs compared to small EVs from the DU145 cells. In green, miRNAs with a log2FC > 1 or <−1 are indicated. In red, miRNAs with an adjusted p value < 0.05. F Log2 Fold Change of miRNAs expressions in DU145 RR-derived small EVs compared to DU145-derived small EVs measured by RT-qPCR (n > 4).

Fig. 2. DNA methylation levels of miR-200c-3p promoter reflect its expression.

A DNA methylation levels of the MIR200/141 promoter in DU145 parental cells, cells irradiated for a total of 60 Gy undergoing radioresistance acquisition, and radioresistant cells, measured by pyrosequencing. CpG sites are represented according to the beginning of the pre-miR-200c sequence. Kruskal–Wallis: *p < 0.05. n = 3. B Average levels of DNA methylation on the MIR200/141 promoter in the small EVs derived from DU145 RR and parental cells measured by methyl-qPCR.

Fig. 3. miR-200c-3p decreases radiation-resistant cell survival by delaying DNA repair.

A Radiation clonogenic survival assays were performed on radioresistant DU145 (DU145 RR) cells transiently transfected with control (miR-neg) or miR-200c-3p mimic. Means and SEM are represented. Wilcoxon test one side at 6 Gy DU145 RR vs DU145+miR-200c-3p or vs DU145: ***p < 0.001, n > 4, RPF radiation protection factor. B Viability test of DU145 radioresistant cells transfected with control or miR-200c-3p mimic. Twenty-four hours after the transfection, the cells were irradiated with 6 Gy before measuring the viability of the cells 24 hours later with a Cell-Titer Glo kit. The luminescence was expressed relative to that in the miR-neg condition. Wilcoxon test one side, **p < 0.01, n = 5. C Representative images of confocal microscopy images for the detection of γ-H2AX foci and 53BP1 foci. Nuclei were stained with Hoechst (blue). In green, the phosphorylation of S139 (γ-H2AX) of histone H2AX, in red 53BP1 protein, in DU145 RR cells transfected with miR-neg or miR-200c-3p, at 30 min, 6 h or 24 h after irradiation at 6 Gy. White bar = 10 μm. D Plot of the number of γ-H2AX foci number per cell. For each condition, more than 50 cells were analyzed per replicat, n = [5–7], Wilcoxon test one side, *p < 0.05. E Plot of the number of 53BP1 foci number per cell. For each condition, more than 50 cells were analyzed per replicat, n = [4–5], Wilcoxon test one side, *p < 0.05.

Fig. 4. Radiation-resistant and radiation-sensitive PCa cells have distinct transcriptomes.

A Volcano plot of differentially expressed mRNAs in DU145 RR cells compared to DU145 cells. The red dots on the left represent significantly downregulated mRNAs in DU145 RR cells, and those on the right represent upregulated mRNAs compared to those in DU145 cells. In green, mRNAs with a log2FC > 0.5 or <-0.5 are shown. In red, mRNAs with an adjusted p value < 0.05. B Volcano plot of differentially expressed mRNAs in DU145 RR cells transfected with miR-200c-3p versus those transfected with a control miRNA. The red dots on the left represent significantly downregulated mRNAs in DU145 RR cells transfected with miR-200c-3p, and those on the right represent upregulated mRNAs compared to those in cells transfected with control miRNA. In green, mRNAs with a log2FC > 0.5 or <−0.5 are shown. In red, mRNAs with an adjusted p value < 0.05. C Top8 of KEGG pathway enrichment of the DU145 RR-deregulated mRNAs D Top8 of KEGG pathway enrichment of the miR-200c-3p transfected DU145 RR-deregulated mRNAs. E Upset plot showing intersections between mRNAs predicted to be miR-200c-3p targets through algorithms (predict), mRNAs upregulated in radioresistant cells (RR), and mRNAs downregulated in DU145 RR cells transfected with miR-200c-3p (miR). Right, list of the 19 mRNAs common to all three groups.

Fig. 5. miR-200c-3p directly targets CBX5 mRNA.

A Relative expression of luminescence of HEK-293 cells transfected with 66 nM of miR-200c-3p (grey) or control miRNA (miR-neg) (white) and WT or mutated constructs in the miR-200c-3p binding site (20 ng). Luminescence expression is relative to the luminescence of miR-neg transfected cells. n = [4–5], Wilcoxon test one side, *p < 0.05. A schematic representation of the wild-type and mutated constructs in the miR-200c-3p-binding site is shown. WT, wild type. B Quantification of HP1α expression in DU145 RR cells transfected with miR-200c-3p or control miRNA (miR-neg) by western blot analysis. The optical density of each sample was measured and normalized using a nucleus housekeeping protein (H3) run on the same gel using ImageJ. The data are expressed as relative expression (HP1α/H3). n = 3, Wilcoxon test, *p < 0.05.

Fig. 6. Silencing of miR-200c-3p is associated with radiation resistance in other cancers.

A miR-200c-3p expression in lung cancer cell lines. The expression of miR-200c-3p is expressed relative to snord44, an endogenous housekeeping RNA. Means and SEM of n = 4 experiments per cell line are represented. B Relationships between radioresistance at 6 Gy and miR-200c-3p expression in lung cancer cell lines. Lung cancer cell lines were grouped according to their miR-200c-3p expression. Means and SEM of n = 3 experiments per cell line are shown. Wilcoxon test. C miR-200c-3p expression in breast cancer cell lines. The expression of miR-200C-3p is expressed relative to snord44, an endogenous housekeeping RNA. Means and SEM of n = 4 experiments per cell line are represented. D DNA methylation levels of the MIR200/141 promoter in lung and breast cancer cell lines measured by pyrosequencing. CpG sites are represented according to the beginning of the pre-miR-200c sequence. E Survival fraction at 6 Gy of A549 and ADCA72 cell lines transfected with miR-200c-3p or a control miRNA. 9 experiments were performed for each cell line. Wilcoxon test. F RT-qPCR of CBX5 mRNA in A549 and ADCA72 cell lines transfected with miR-200c-3p compared to a control miRNA. n = 7 experiments per cell line. *p < 0.05, **p < 0.01, ***p < 0.001.