Retracted Article: Gemcitabine aggravates miR-199a-5p-mediated breast cancer cell apoptosis by promoting VEGFA downregulation via inactivating the AKT signaling pathway

Abstract

Breast cancer is the most frequent malignancy diagnosed in women, and Gemcitabine-based therapy is frequently used to treat late-stage breast cancer. miR-199a-5p plays a tumor-suppressive role in breast cancer. This work aimed to explore the mechanism of miR-199a-5p plus Gemcitabine in breast cancer cells. Expression of miR-199a-5p was measured by RT-qPCR, while expression of vascular endothelial growth factor A (VEGFA) was measured by Western blot and RT-qPCR. Overexpression of miR-199a-5p and/or silencing of VEGFA was obtained using transfection in breast cancer cells (MCF-7 and MDA-MB-231). Functional experiments were performed to explore cell viability, apoptosis rate, and expressions of apoptosis-related genes: cell viability was assessed by MTT staining, apoptosis rate was recorded by flow cytometry, and Western blot was used to evaluate the expressions of Bcl-2, Bax and cleaved caspase 3. The signaling pathway was studied with respect to AKT activity via determination of p-AKT expression levels. Our study found that miR-199a-5p was downregulated and VEGFA was upregulated in breast cancer tissues and cells. Overexpression of miR-199a-5p and/or silencing of VEGFA contributed to cell apoptosis and inhibited cell viability, which was promoted by Gemcitabine. VEGFA was a downstream target of miR-199a-5p, and was negatively regulated by Gemcitabine. Moreover, Gemcitabine aggravated the miR-199a-5p-induced suppression of the VEGFA level and AKT activity in breast cancer cells. Our data show that Gemcitabine aggravates miR-199a-5p-mediated VEGFA downregulation and apoptosis via inactivating the AKT signaling pathway in breast cancer cells, indicating a novel promising combined therapy of miR-199a-5p overexpression and Gemcitabine.

Fig. 1. The role of miR-199a-5p in breast cancer. (A and B) Expression of miR-199a-5p in breast cancer tissues and cell lines (MCF-7 and MDA-MB-231) was measured with RT-qPCR, compared with normal tissue and the cell line MCF-10A. (C) The overexpression of miR-199a-5p/NC mimic (miR-199a-5p/NC) in breast cancer cells using transfection and expressions of miR-199a-5p were detected. (D and E) Cell viability and apoptosis were separately assessed by MTT staining and flow cytometry in cells (control) and cells transfected with miR-199a-5p/NC. (F) Western blot detected the expressions of apoptosis-related genes, Bcl-2, Bax and cleaved caspase 3 (cle-caspase 3). All experiments were performed in triplicate and *P < 0.05.

Fig. 2. Gemcitabine aggravated miR-199a-5p overexpression-induced cell apoptosis in breast cancer cells. MCF-7 and MDA-MB-231 cells were treated with transfection of miR-199a-5p/NC mimic and Gemcitabine or not. (A) Cell viability was measured. miR-199a-5p cells had reduced OD490 values, and a combination of miR-199a-5p and Gemcitabine resulted in even lower OD490 values (^&P < 0.05) than treatment by miR-199a-5p overexpression only. (B) The apoptosis rate was recorded. miR-199a-5p cells had raised apoptotic cell counts, while the combination of miR-199a-5p and Gemcitabine resulted in even higher numbers of apoptotic cells (^&P < 0.05) than treatment by miR-199a-5p overexpression only. (C) Expressions of Bcl-2, Bax and cle-caspase 3 were detected. (D) The effect of Gemcitabine on cell viability in cells overexpressing miR-199a-5p/NC. Cell viability was impaired by Gemcitabine, and *P < 0.05. All experiments were performed in triplicate.

Fig. 3. Gemcitabine aggravated VEGFA silencing-induced breast cancer cell apoptosis. (A and B) Expressions of VEGFA in breast cancer tissues and cell lines (MCF-7 and MDA-MB-231) were measured with RT-qPCR. VEGFA was upregulated in breast cancer compared with normal tissue and the cell line MCF-10A. (C) Expression levels of VEGFA mRNA were detected in breast cancer cells when transfected with siRNA against VEGFA/scramble (siVEGFA/scramble). (D and E) Cell viability and apoptosis were separately assessed by MTT staining and flow cytometry in cells cultured with siVEGFA/scramble and Gemcitabine or not. (F) Western blot detected the expressions of Bcl-2, Bax and cle-caspase 3 in siVEGFA/scramble-transfected cells with treatment using Gemcitabine or not. All experiments were performed in triplicate and *P < 0.05.

Fig. 4. VEGFA expression was negatively regulated by miR-199a-5p directly binding in MCF-7 cells. (A) A prediction of the potential binding sites between hsa-miR-199a-5p and VEGFA and the construction of VEGFA 3′ UTR wild type and mutant (VEGFA-wt/mut). (B and C) Luciferase assay and RNA immunoprecipitation (RIP) confirmed this to be the target binding site in MCF-7 cells. (D) Regulatory effects of miR-199a-5p on VEGFA expression were measured with Western blot. The gray intensity was calculated with normalization to the control (MCF-7 cells without transfection). All experiments were carried out in triplicate and *P < 0.05.

Fig. 5. Gemcitabine promoted miR-199a-5p-mediated VEGFA downregulation in breast cancer cells. (A) VEGFA protein expression was inversely regulated by Gemcitabine incubation. The gray intensity was calculated with normalization to cells without Gemcitabine treatment. (B) The effect of a combination of miR-199a-5p and Gemcitabine on VEGFA protein expression. The gray intensity was calculated with normalization to the control (cells without transfection or Gemcitabine treatment). *P < 0.05. All experiments were carried out in triplicate.

Fig. 6. The AKT signaling pathway was inactivated by a combination of miR-199a-5p and Gemcitabine through VEGFA downregulation in breast cancer cells. AKT activation was evaluated from p-AKT expression according to Western blot. (A) The effect of VEGFA silencing on p-AKT expression. (B) The effects of a combination of miR-199a-5p and Gemcitabine on p-AKT expression. *P < 0.05. All experiments were carried out in triplicate.