Combination therapy with miR34a and doxorubicin synergistically inhibits Dox-resistant breast cancer progression via down-regulation of Snail through suppressing Notch/NF- κ B and RAS/RAF/MEK/ERK signaling pathway

Abstract

Resistance to breast cancer (BCa) chemotherapy severely hampers the patient's prognosis. MicroRNAs provide a potential therapeutic prospect for BCa. In this study, the reversal function of microRNA34a (miR34a) on doxorubicin (Dox) resistance of BCa and the possible mechanism was investigated. We found that the relative level of miR34a was significantly decreased in Dox-resistant breast cancer cell MCF-7 (MCF-7/A) compared with Dox-sensitive MCF-7 cells. Transfection with miR34a significantly suppressed the invasion, migration, adhesion of MCF-7/A cells without inhibiting their growth obviously. The combination of miR34a and Dox could significantly inhibit the proliferation, migration, invasion and induce the apoptosis of MCF-7/A cells. The synergistic effect of this combination on resistant MCF-7/A cells has no obvious relation with the expressions of classical drug-resistant proteins P-GP, MRP and GST-π, while closely related with the down-regulation on TOP2A and BCRP. Moreover, we found both protein and mRNA expression of Snail were significantly up-regulated in MCF-7/A cells in comparison with MCF-7 cells. Transfection with small interfering RNA (siRNA) of Snail could inhibit the invasion, migration and adhesion of drug-resistant MCF-7/A cells, while high-expression of Snail could remarkably promote the invasion, migration and adhesion of MCF-7 cells, which might be related with regulation of N-cadherin and E-cadherin. Transfection with miR34a in MCF-7/A cells induced a decrease of Snail expression. The potential binding sites of miR34a with 3' UTR of Snail were predicted by miRDB target prediction software, which was confirmed by luciferase reporter gene method. Results showed that the relative activity of luciferase was reduced in MCF-7/A cells after co-transfection of miR34a and wild type (wt)-Snail, while did not change by co-transfection with miR34a and 3' UTR mutant type (mut) Snail. Combination of miR34a and Dox induced a stronger decrease of Snail in MCF-7/A cells in comparison to miR34a or Dox treatment alone. What' more, for the first time, we also found miR34a combined with Dox could obviously inhibit the expression of Snail through suppressing Notch/NF-κB and RAS/RAF/MEK/ERK pathway in MCF-7/A cells. In vivo study indicated that combination of miR34a and Dox significantly slowed down tumor growth in MCF-7/A nude mouse xenograft model compared with Dox alone, which was manifested by the down-regulation of Snail and pro-apoptosis effect in tumor xenografts. These results together underline the relevance of miR34a-driven regulation of Snail in drug resistance and co-administration of miR34a and Dox may produce an effective therapy outcome in the future in clinic.

Keywords: Breast cancer; Dox; Drug resistance; Notch/NF-κB; RAS/RAF/MEK/ERK; Snail; Therapy; miR34a.

Graphical abstract

Figure 1

Differences between Dox-sensitive MCF-7 cells and Dox-resistant MCF-7/A cells. (A) Wound scratch assay was employed to compare the migration difference between MCF-7 and MCF-7/A cells. (B) Transwell assay was used to compare the invasion and migration ability between MCF-7 and MCF-7/A cells. Adhesion ability of MCF-7 or MCF-7/A cells with HUVEC cells was detected by adhesion assay. (C) Expression differences of EMT-related proteins (N-cadherin, E-cadherin, CD44) between the MCF-7 and MCF-7/A cells were determined by Western blotting assay. Scale bar = 20 μm.

Figure 2

Transfection of miR34a inhibited the invasion, migration, and adhesion to HUVECs of MCF-7/A cells. (A) mRNA level of miR34a in MCF-7/A cells or MCF-7 cells was detected by qRT-PCR. (B) mRNA level of miR34a after transfection with miR34a mimics at different concentrations in MCF-7/A cells was detected by qRT-PCR. (C) Effects of miR34a at different concentrations on the proliferation were determined by MTT assay in MCF-7/A cells. (D) Effects of miR34a at different concentrations on the invasion, migration and adhesion to HUVECs of MCF-7/A cells were detected. (E) The migration of MCF-7/A cells after transfection with miR34a at 100 nmol/L was detected by wound scratch test. (F) The expression levels of EMT-related proteins (N-cadherin, E-cadherin, CD44) after transfection with miR34a for 48 h in MCF-7/A cells were detected by Western blotting assay and the relative protein ratios were analyzed statistically (n ≥ 3). ∗P < 0.05, scale bar = 20 μm.

Figure 3

The combination of miR34a and Dox significantly suppressed the survival and induced the apoptosis in MCF-7/A cells. (A) The IC₅₀ values of Dox on MCF-7/A cells when Dox is used alone or in combination with different concentrations of miR34a (25, 50, and 100 nmol/L) for 48 h were determined. (B) The combination of 100 nmol/L miR34a and 10 μmol/L Dox remarkably suppressed the survival of MCF-7/A cells compared with Dox alone (n = 3). ∗∗∗P < 0.001 vs. untreated MCF-7/A, #P < 0.05 vs. Dox alone. (C) Hoechst 33342 was used to detect the effects of 100 nmol/L miR34a alone, 10 μmol/L Dox alone or a combination on MCF-7/A cell apoptosis after treatment for 48 h. Blue indicates the nucleus. Arrows indicate apoptotic cells. Scale bar = 20 μm. (D) After treatment with miR34a or Dox alone or co-administration for 48 h, MCF-7/A cells were double-stained with Annexin V-FITC/PI kit and analyzed with flow cytometer. (E) Protein levels of cleaved-PARP, BAX, BCL-2 in MCF-7/A cells were analyzed by Western blotting. (F) Quantitative analysis of apoptosis rate which included early and late apoptotic cells (n ≥ 3). #P < 0.05 vs. Dox or miR34a alone. (G) The relative protein ratio of cleaved-PARP and the ratio of BCL-2/BAX were calculated (n ≥ 3). ∗P < 0.05, ∗∗P < 0.01 vs. untreated MCF-7/A cells. miR represents miR34a.

Figure 4

Effects of miR34a and Dox co-administration on the expression of drug resistant associated proteins. (A) The expressions of P-GP, GST-π, MRP, BCRP and TOP2A in MCF-7/A cells were determined by Western blotting assay. (B–F) Statistical analysis of P-GP (B), GST-π (C), MRP (D), TOP2A (E) and BCRP (F). Data are set as the mean value ± SD (n ≥ 3). ∗P < 0.05, ∗∗P < 0.01, vs. untreated MCF-7/A cells. miR represents miR34a.

Figure 5

Combination of miR34a and Dox inhibited the migration and invasion phenotype of MCF-7/A via regulating the level of E-cadherin, N-cadherin. (A) After treatment with miR34a (100 nmol/L) and/or Dox (7 μmol/L), scratch test was employed to detect the changes of migration in MCF-7/A cells. Representative photographs for each group were taken under a microscopy, and the migration distance and relative mobility were calculated at 0, 24 and 48 h. (B) Statistical analysis of relative migration rate which was calculated as (width at 0 h−width at 24/48 h)/(width at 0 h) (n = 3), ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 vs. untreated MCF-7/A control, #P < 0.05 vs. Dox alone. (C) The migration and invasion capacities of MCF-7/A cells after treatment of miR34a (Dox) alone and combination were assessed by Transwell chamber assay. Representative photographs were taken under a fluorescence microscope. (D) Statistical analysis of relative migration and invasive cells (n = 3). ∗∗P < 0.01, ∗∗∗P < 0.001 vs. untreated MCF-7/A control, #P < 0.05 vs. Dox or miR34a alone, ##P < 0.01 vs. Dox alone. (E) The protein levels of N-cadherin, E-cadherin were detected using Western blotting analysis. (F) Statistical analysis of protein expression. Data are expressed as the mean ± SD (n ≥ 4). ∗P < 0.05 vs. untreated MCF-7/A control, #P < 0.05 vs. Dox or miR34a alone, scale bar = 20 μm, miR represents miR34a.

Figure 6

Combination of miR34a and Dox inhibited Snail expression and miR34a targets Snail directly in MCF-7/A cells. (A) The intracellular localization of Snail was analyzed using immunofluorescence experiments. Blue fluorescence: cell nucleus, Green fluorescence: Snail. Bar = 20 μm. (B) The expression of Snail in the two cells was determined and analyzed by Western blotting (n ≥ 3). ∗P < 0.05 vs. untreated MCF-7/A control, #P < 0.05 vs. miR34a alone. (C) The expression of Snail in MCF-7/A cells after various treatments was determined and analyzed by Western blotting (n ≥ 3). ∗P < 0.05 vs. Dox group, ∗∗P < 0.01 vs. Dox+NC group. (D) Binding sites between hsa-miR34a-5p and Snail were predicted by miRDB website. (E) and (F) The protein expression and mRNA level of Snail in MCF-7/A cells after transfection with miR34a and NC were detected by Western blotting and qRT-PCR (n ≥ 3). ∗P < 0.05 vs. NC. (G) Mutant and wild type Snail fragments with putative binding sites to miR34a were constructed. (H) The binding of miR34a and Snail was determined by luciferase reporter assay using co-transfection of miR34a/NC and wt-Snail, or miR34a/NC and mut-Snail. The ratio of Firefly and Renilla luciferase signals represented relative luciferase activity. ∗P < 0.05 vs. NC. miR represents miR34a.

Figure 7

Knock down of Snail by siRNA inhibited the invasion, migration and adhesion to HUVECs of MCF-7/A cells while over-expression of Snail promoted the invasion, migration and adhesion of MCF-7 cells. BC: Blank control group, NC: Negative control group, si-Snail: Snail siRNA group, Snail: Snail over-expression group. (A) Cell viability of MCF-7/A cells cultured with si-Snail for 48 h was tested by MTT assay (n = 3). ∗P < 0.05 vs. NC. (B) The protein levels of N-cadherin, E-cadherin and Snail in MCF-7/A cells treated with si-Snail for 48 h were determined by Western blotting. (C) and (J) Statistical analysis of E-cadherin, N-cadherin, CD44 and Snail proteins was performed. ∗P < 0.05 vs. BC or NC, #P < 0.05, &P < 0.05 vs. NC. (D) The migration of MCF-7/A cells after incubation with si-Snail for 48 h was measured by wound scratch assay. (E) Transwell assay and cell adhesion assay were further employed to examine the migration, invasion and adhesion of MCF-7/A cells after incubation with Snail siRNA, respectively. (F) The protein level of Snail in MCF-7/A cells cultured with si-Snail for 48 h was observed by immunofluorescence assay. (G) The mRNA level of Snail in MCF-7 cells was determined by qRT-PCR. ∗∗P < 0.01 vs. BC, ##P < 0.01 vs. NC. (H) The cell survival rate after over-expression of Snail in MCF-7 cells was detected by MTT assay (n = 3). “ns” indicates no significance. (I) The expressions of Snail, N-cadherin and E-cadherin in MCF-7 cells were examined by Western blotting. (K) The migration of MCF-7 cells after over-expression of Snail was observed by scratch assay. (L) The invasion, migration and adhesion abilities of MCF-7 cells were detected by Transwell assay and adhesion assay. Bar = 20 μm. (M) Snail expression in MCF-7 cells was detected by immunofluorescence assay.

Figure 8

Co-administration of miR34a and Dox down-regulated Notch/NF-κB and RAS/RAF/MEK/ERK pathway in MCF-7/A cells. (A) The protein expressions of Notch and NF-κB were determined by Western blotting. (B) The levels of pan-RAS, k-RAS, RAF, MEK1/2, ERK, p-MEK1/2, p-ERK were determined by Western blotting assay. (C) Statistical analysis of protein expression of Notch, NF-κB, MEK1/2, p-MEK1/2, RAF, p-ERK, ERK, pan-RAS and k-RAS was performed (n ≥ 3). ∗P < 0.05, ∗∗P < 0.01 vs. Untreated MCF-7/A control. #P < 0.05 vs. Dox alone. (D) Effects of Notch inhibitor DAPT, NF-κB inhibitor PDTC, k-RAS inhibitor salirasib and MEK inhibitor pimasertib at different concentrations on the pathway associated protein expression were determined by Western blotting assay (n ≥ 3). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001; #P < 0.05; &P < 0.05. miR represents miR34a.

Figure 9

Combination of agomiR34a and Dox slowed down the xenograft growth of MCF-7/A cells in vivo. (A) Representative images of xenografts after administration of agomiR34a, Dox or agomiR34a combined with Dox for 21 days. (B) and (C) Body weight and tumor volume were determined every three days for 21 days (n = 3). ∗∗∗P < 0.001 vs. Control. (D) After the experiment was over, nude mice were sacrificed and the tumor tissues were weighed. ∗P < 0.05 vs. Dox, #P < 0.05 vs. AgomiR34a. (E) The protein expressions of Snail, BCL-2, BAX and cleaved PARP in MCF-7/A tumor tissues were detected and analyzed using Western blotting (n ≥ 3). (F) Statistical analysis of relative expression of cleaved PARP, Snail and the ratio of BCL-2/BAX was performed. ∗P < 0.05 vs. Dox, #P < 0.05 vs. AgomiR34a. (G) Expressions of Snail in tumor xenograft were analyzed by immunofluorescence assay. (H) H&E staining and immunohistochemical assay were performed for tumors in different groups. Scale bar = 20 μm.

Figure 10

The proposed pathway of synergistic anti-tumor effects of miR34a and Dox in MCF-7/A cells.