Fig 1.
MiR-202-3p is downregulated in brain metastatic breast cancer cell lines and varies inversely with MMP-1 expression.
(A) MiR-202-3p relative expression was measured by real-time PCR in three breast cancer cell lines with different brain metastatic propensities (MCF-7; MDA-MB-231-TGL and MDA-MB-231-BrM2). The small nuclear RNA (RNU6-2) was used as an internal standard. Data are represented as 2^-ΔCt. (B-D) The transmigrative ability of BC cells through a monolayer of brain endothelial cells was assessed by trans-endothelial migration assay. Cancer cells were labelled with the CellTracker™Green CMFDA fluorescent dye and added on (B) primary brain endothelial cells or (C) hCMEC/D3 monolayer. Four hours later, non-adherent cells were removed by PBS washing. Twenty-four hours following the co-culture, cancer cells that transmigrated to the lower chamber were lyzed with RIPA buffer and fluorescent intensity was measured at excitation/emission: 492/517 nm. Results are expressed as fold change of the fluorescence intensity (FI) relative to MCF-7. The average percentage of plated cells that transmigrated through the layer of primary brain endothelial cells was around 1% for MCF7, 4% for MDA-MB-231-TGL and 15% for MDA-MB-231-BrM2. The average percentage of plated cells that transmigrated through the hCMEC/D3 layer was around 3% for MCF7, 11% for MDA-MB-231-TGL and 42% for MDA-MB-231-BrM2. (D) Cancer cells that transmigrated through the hCMEC/D3 monolayer and the pores of the filter were counted after 24 hours of co-culture in three different fields per insert under fluorescent microscope. Results are expressed as fold change of transmigrated cell number relative to MCF-7. Representative images of transmigrated fluorescently labelled breast cancer cells are shown. The average number of transmigrating cells per field of vision (FOV) ranged from 0–1 for MCF-7, 2–4 for MDA-MB-231-TGL and 8–20 for MDA-MB-231-BrM2. (E) The relative mRNA expression of MMP1 was measured by real-time PCR. GAPDH was used as an internal standard. Data are represented as 2^-ΔCt. (F, G) The protein expression of MMP-1 was examined by western-blot (F) and immunofluorescence (G). Optical densities of three independent images were analyzed with Image Lab 6.0.1 software(Bio-Rad) and normalized to GAPDH. Results are represented as fold change of normalized optical densities relative to MCF-7. Experiments were carried out three to four times. Data represent mean ± SD. Scale bar = 50 μm. *p<0.05, **p<0.01, ***p<0.001.
Fig 2.
MiR-202-3p directly targets the 3’UTR of MMP1.
(A) Schematic representation of the MMP1 3’-UTR with the miR-202-3p binding site. Complementary sequences are represented in blue. (B,C) Effect of miR-202-3p (wild type and mutant) on MMP1 3’UTR luciferase reporters. Constructs carrying MMP1 3’UTR luciferase reporter (pmirGLO-MMP1-3’UTR-WT) or deletion mutant of miR-202-3P binding site (pmirGLO-MMP1-3’UTR-MUT) were co-transfected with miR-202-3p mimic (or negative control) into (B) MDA-MB-231 (ATCC) and (C) MDA-MB-361 cells and subjected to luciferase assays after twenty-four hours. Results are expressed as the fold change of the ratio (firefly to Renilla luciferase activity). Experiments were carried out three to four times. Data represent mean ± SD. *p<0.05, **p<0.01, ***p<0.001.
Fig 3.
MiR-202-3p suppression enhances MMP-1 expression in breast cancer cells.
MDA-MB-231-TGL cells were transiently transfected with miR-202-3p inhibitor or negative control. (A) After 48h, total RNA was extracted and miR-202-3p relative expression was measured by real-time PCR. The small nuclear RNA (RNU6-2) was used as an internal standard. Data are represented as 2^-ΔCt. (B) The relative mRNA expression of MMP1 was measured by real-time PCR. GAPDH was used as an internal standard. Data are represented as fold change (2^-ΔΔCt) of normalized MMP1 mRNA levels relative to the negative control. (C, D) The protein expression of MMP-1 was examined by western-blot (C) and immunofluorescence (D). Optical densities of three independent images were analyzed with Image Lab 6.0.1 software(Bio-Rad) and normalized to GAPDH. Results are represented as normalized optical densities. (E) MMP-1 levels released in the culture media were quantified by ELISA. Results are represented as fold change of MMP1 concentration levels relative to the negative control. (F) Cells were co-transfected with miR-202-3p and MMP1 siRNA and protein expression of MMP1 was examined by western-blot. Optical densities of three independent images were analyzed with Image Lab 6.0.1 software (Bio-Rad) and normalized to GAPDH. Results are represented as normalized optical densities. Experiments were carried out three to six times. Data represent mean ± SD. Scale bar = 50 μm. *P<0.05, **P<0.01, ***P<0.001.
Fig 4.
Ectopic downregulation of miR-202-3p in breast cancer cells enhances their transmigration through the brain endothelium and disrupts the endothelial barrier integrity.
MDA-MB-231-TGL cells were transfected with miR-202-3p inhibitor (30 nM final concentration) or with negative control. (A,B) The transmigration abilities of the different BC cells were examined by trans-endothelial migration assay. Transfected cancer cells were labelled with the CellTracker™Green CMFDA fluorescent dye and added on (A) primary BECs or on (B) hCMEC/D3 monolayers. Cancer cells that transmigrated to the lower chamber were lyzed with RIPA buffer and fluorescent intensity was measured at excitation/emission: 492/517 nm. Results are expressed as fold change of fluorescent intensity relative to negative control. The average percentage of plated MDA-MB-231-TGL cells that transmigrated through the layer of primary brain endothelial cells was around 4% of control cells and 9% of transfected cells. The average percentage of plated cells that transmigrated through the hCMEC/D3 layer was around 10% of control cells and 27% of transfected cells. (B) Transfected cancer cells that transmigrated through the hCMEC/D3 monolayer and the pores of the filter were counted after 24 hours in three different fields per insert under fluorescent microscope. Results are expressed as fold change of transmigrated cell number relative to MCF-7. Representative images of transmigrated fluorescently labelled MDA-MB-231-TGL (small green cells) in control and anti-miR-202-3p transfected cells are shown. The average number of transmigrating cells per field of vision (FOV) ranged from 1–3 for control cells and 5–10 for transfected cells. (C) Transendothelial electrical resistance (TEER) was measured for brain endothelial cells co-cultured with MDA-MB-231-TGL cells treated with miR-202-3p inhibitor or negative control after 24 hours of co-culture. (D) Protein expression of the inter-endothelial junctions (ZO-1, ß-catenin and claudin-5) was measured by western-blot in hCMEC/D3 co-cultured with MDA-MB-231-TGL cells treated with miR-202-3p inhibitor or control. (E) Fold change of the fluorescent intensity of transmigrated MDA-231-TGL cells pre-transfected with miR-202-3p inhibitor and/or MMP1 SiRNA or negative control. (F) TEER values of hCMEC/D3 cells co-cultured with MDA-MB-231-TGL cells treated with miR-202-3p inhibitor and/or MMP1 SiRNA or negative control after 24 hours of co-culture. (G) Protein expression of the inter-endothelial junctions (ZO-1, ß-catenin and claudin-5) protein expression measured by western-blot in hCMEC/D3 co-cultured with MDA-MB-231-TGL cells treated with miR-202-3p inhibitor and/or MMP1 siRNA or negative control. Optical densities of three independent images were analyzed with Image Lab 6.0.1 software (Bio-Rad) and normalized to GAPDH. Data are mean ±SD from three independent experiments. *p<0.05, **p<0.01, ***p<0.001.
Fig 5.
MiR-202-3p upregulation downregulates MMP1 expression in brain metastatic breast cancer cells.
MDA-MB-231-BrM2 cells were transiently transfected with miR-202-3p mimic or scrambled control. (A) After 48h, total RNA was extracted and miR-202-3p relative expression was measured by real-time PCR. The small nuclear RNA (RNU6-2) was used as an internal standard. Data are represented as fold change (2^-ΔCt) of normalized miR-202-3p levels relative to the control (scrambled). (B) The relative mRNA expression of MMP1 was measured by real-time PCR. GAPDH was used as an internal standard. Data are represented as fold change (2^-ΔΔCt) of normalized MMP1 mRNA levels relative to the negative control. (C, D) The protein expression of MMP-1 was examined by western-blot (C) and immunofluorescence (D). Optical densities of three independent images were analyzed with Image Lab 6.0.1 software (Bio-Rad) and normalized to GAPDH. Results are represented as normalized optical densities. (E) MMP-1 levels released in the culture media were quantified by ELISA. Results are represented as fold change of MMP1 concentration levels relative to the control (scrambled). Experiments were carried out three times. Data represent mean ± SD. Scale bar = 50 μm. *p<0.05, **p<0.01 and ***p<0.001.
Fig 6.
Ectopic upregulation of miR-202-3p in breast cancer cells suppresses their transmigration through the brain endothelium and preserves the endothelial barrier integrity.
MDA-MB-231-BrM2 cells were transfected with miR-202-3p mimic or scrambled control. (A-C) The transmigration abilities of the different BC cells were examined by trans-endothelial migration assay. Transfected cancer cells were labelled with the CellTracker™Green CMFDA fluorescent dye and added on (A) primary BECs or (B) hCMEC/D3 monolayer. Results are expressed as fold change of fluorescent intensity relative to the control (scrambled). The average percentage of plated MDA-MB-231-BrM2 cells that transmigrated through the layer of primary brain endothelial cells was around 18% of control cells and 6% of transfected cells. The average percentage of plated cells that transmigrated through the hCMEC/D3 layer was around 39% of control cells and 9% of transfected cells. (C) Transfected cancer cells that transmigrated through the endothelial monolayer and the pores of the filter were counted after 24 hours in three different fields per insert under fluorescent microscope. Results are expressed as fold change of transmigrated cell number relative to MCF-7. Representative images of transmigrated fluorescently labelled MDA-MB-231-BrM2 (small green cells) in control and miR-202-3p transfected cells are shown. The average number of transmigrating cells per field of vision (FOV) ranged from 8–14 for control cells and 2–5 for transfected cells. (D) Trans-endothelial electrical resistance (TEER) was measured for brain endothelial cells co-cultured with MDA-MB-231-BrM2 cells treated with miR-202-3p mimic or scrambled control. (E) Western-blot analysis of the inter-endothelial junctions (ZO-1, ß-catenin and claudin-5) protein expression was measured by western-blot in hCMEC/D3 co-cultured with MDA-MB-231-BrM2 cells treated with miR-202-3p mimic or scrambled control. Optical densities of three independent images were analyzed with Image Lab 6.0.1 software (Bio-Rad) and normalized to GAPDH. Results are represented as fold change of normalized optical densities relative to the control (scrambled). Data are mean ±SD from three to five independent experiments. *p<0.05, **p<0.01, ***p<0.001.