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MicroRNA-454 may function as an oncogene via targeting AKT in triple negative breast cancer
© The Author(s) 2017
Received: 5 April 2017
Accepted: 20 July 2017
Published: 3 August 2017
Altered microRNAs expression mediates tumor development and progression in many type cancers including triple negative breast cancer (TNBC). Here we detected the effect of miR-454 on cell proliferation, migration and invasion of triple negative breast cancer cells.
miR-454 promoted the proliferation of TNBC, and enhanced migration and invasion in TNBC cells. Meanwhile, miR-454 improved the survival of TNBC cells after ironizing radiation. miR-454 inhibited radiation-induced apoptosis in TNBC cells by regulation of caspase 3/7 and Bcl-2 expression. Furthermore, PTEN and pAKT levels in TNBC cells were changed after overexpression of miR-454.
miR-454 played an essential role in tumor development and progression in TNBC, and might be used as a potential biomarker to predict radiotherapy response and prognosis in TNBC.
Breast cancer remained the most commonly diagnosed cancer in women and the second leading cause of cancer death among women . In the past decade, early detection and screening and improvement in medical care and treatments decreased dramatically the mortality of patient with breast cancer [2, 3].
Triple-negative breast cancer (TNBC) is a subtype of breast cancer characterized as negative for estrogen receptors, progesterone receptors, and HER2 . Studies have demonstrated that TNBC is an aggressive disease with unique molecular profile . Brain and lungs are the most common metastasis sites of TNBC . There are no targeted therapies for TNBC. Therefore, it is important to discovery of new molecular targets to treat patients with TNBC [7, 8].
It is well known that microRNAs (miRNAs) play critical roles in regulation of gene expression . Studies have demonstrated that miRNAs can behave as both oncogenes and tumor suppressor genes . MiRNAs regulate many biological processes, such as cell proliferation, apoptosis and cell cycle. Several studies have shown that miR-10b improves cell migration and invasion by targeting HOXD10 [11, 12]. Huang et al. has reported that miR-21 is associated with breast cancer cell invasion and regulated epithelial-to-mesenchymal transition (EMT) . Han et al.  has shown that miR-21 regulates breast cancer stem cell-like cells.
Zhang et al. has reported that miR-155 improves breast cancer cell proliferation by regulation of p53 . miR-125b is downregulated in breast cancer and correlates with metastasis and HER2 expression . Recent studies indicate that miRNAs are associated with prognosis in breast cancer  while miR-454 plays critical roles in tumor development in many organs [18, 19]. Zhu et al. found that miR-454 promoted proliferation of non-small cell lung cancer cells by targeting PTEN . The oncogene role of miR-454 is also found in uveal melanoma . Yu et al. reported that miR-454 enhanced hepatocellular carcinoma cell proliferation, invasion and epithelial mesenchymal transition . Liu et al.  and Liang et al.  showed that miR-454 regulated cell proliferation in colon cancer by different signaling pathways.
In this study, we examined the function of miR-454 on cell proliferation with or without radiation, invasion and migration using triple-negative breast cancer cell lines.
Cell lines and cell culture
Two human triple-negative breast cancer cell lines, MDA-MB-231 and MDA-MB-468 (ATCC, USA), were used in all experiments in our study in order to ensure all data are not cell-line restricted. These tumor cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen, USA) supplemented with 100 µg ml−1 streptomycin, 100 U ml−1 penicillin and 10% fetal bovine serum (Invitrogen, USA) at 37 °C in a humidified incubator with 5% CO2. When all cells reach to 80% confluence, they were harvested and used in our experiments.
Cell transfection and miRNA quantification
Lipofectamine 2000 (Invitrogen, USA) was used to transfer miR-454 mimics or anti-miR-454 inhibitor (Invitrogen, USA) into TNBC cells. A random sequence miRNA mimic molecule was used as a negative control (mirVana™miRNA mimic, Ambion, USA). Then, miR-454 expression level was examined by real time PCR after transfection. The total RNA was extracted from the transfected TNBC cells; TaqMan microRNA reverse transcription kit (ThermoFisher, USA) was used to synthesize cDNA according the manufacturer’s instructions. GAPDH was used as the endogenous reference gene.
Cell proliferation assay
WST-1 assay (Roche, USA) was performed to analyze the function of miR-454 on TNBC cell proliferation. Briefly, the transfected TNBC cells were seeded into 96-well plates at the density of 2 × 104 cells well−1. The cells were cultured overnight at 37 °C in a humidified incubator with 5% CO2. Then, the TNBC cells were exposed to different doses of ionizing radiation. Then WST-1 reagent was added to each well and incubated for 1 h at 37 °C every 24 h. Afterwards, the absorbance was measured at 490 nm. All experiments were performed in triplicates.
Migration and invasion assays
The Promega migration and invasion assays were performed to evaluate migration and invasion of TNBC cell following the manufacturer’s instructions (Promega Corporation, USA). Briefly, transfected TNBC cells were placed on the upper transwell chamber either present or absent matrigel in DMEM. DMEM supplemented with 5% FBS was added to the lower chamber. The cells were cultured at 37 °C for 18 h. Then Diff-Quik stain (Invitrogen, USA) was performed after the non-invaded cells were removed by cotton swabs. The migration and invasion was calculated and showed as a ratio of invaded cells over cells normalized on second day of growth curve.
Western blot assay
The TNBC cells were harvested and gently washed with cold phosphate buffered saline. The cells were lysed by adding ice-cold lysis buffer (50 mM Tris–HCl, pH 7.5, 0.1% SDS, 150 mM NaCl, 0.5% deoxycholate, 1% NP-40, and 1× protease inhibitors). The mixture was boiled at 100 °C for 5 min. The same amount of protein lysates were loaded to the SDS-PAGE gel and transferred to PVDF membranes (Sigma, USA). The PVDF membranes were incubated in blocking buffer at room temperature for 1 h. Primary antibodies (Bcl-2, Bax, AKT and pAKT; Cell Signaling Technology, USA) were added in TBS-T buffer and incubated at 4 °C overnight, followed by secondary antibody incubation at room temperature for 1 h. The signals were examined with the EasySeeWeatern Blot Kit (Transgen, Shanghai).
Apoptosis activity assay
To examine the apoptosis activity of TNBC cells with altered miR-454 expression, the TNBC cells were grown in 24-well plates at density of 1 × 105 well−1. Then the TNBC cells were exposed to different doses of ionizing radiation. The TNBC cells were continued to culture for 24 h, the caspase-Glo3/7 assay kit (Promega, Madison, USA) was used to examine apoptosis activity by measure the caspase 3/7 activity following the manufacturer’s protocol. Briefly, caspase-Glo reagent was added to the TNBC cells, and incubated at room temperature in a dark place with gentle shaking for 8 h. The luminescence value was measured using 1-min lag time and 0.5 s well−1 read time. All experiments were carried out in triplicates.
SPSS software (version 11.0, IBM, USA) was used for statistical analyses using Student’s t test. Differences are considered statistically significant if p < 0.05.
miR-454 increased the proliferation of TNBC cells and improved the survival at ionizing radiation
miR-454 decreased radiation-induced apoptosis in TNBC cells
miR-454 improved migration and invasion of TNBC cells
miR-454 inhibited PTEN and activated pAKT in TNBC cells
Recent studies have shown that miR-454 is highly expressed in tissue from patients with TNBC . These findings indicate that miR-454 expression level might be positively correlated with worse clinical outcome. Therefore, miR-454 might act as a potential predictor of prognosis in TNBC. Recently, a tissue microarray study showed that miR-454 is associated with poor prognosis in patients with TNBC . In our study, we found that miR-454 improved the proliferation of TNBC cells. Radiotherapy is one of highly effective adjuvant therapies in many types of cancer including breast cancer after surgery . TNBC is a complex disease with features such as rapid growth and local recurrence. It has been reported that radiotherapy can decrease the locoregional recurrence in patients at early stage after surgery . In the present study, we showed that miR-454 improved the survival of TNBC cells after ionizing radiation. Meanwhile, miR-454 inhibited the radiation-induced apoptosis of TNBC cells. Moreover, we showed that overexpression of miR-454 increased migration and invasion in TNBC cells. However, downregulation of miR-454 inhibited the migration and invasion in TNBC cells. It has been demonstrated that TNBC is more likely to recur and has poor prognosis . Metastasis is one of important features for the recurrence of TNBC. Therefore, our study showed that miR-454 play critical roles in TNBC progression.
The PI3K/AKT pathway involves many biological processes and regulates the downstream responses including cell proliferation, apoptosis and cell cycle. It has been reported that PI3K/AKT is activated in TNBC due to PTEN mutation . PTEN, known as the second most frequently mutated tumor suppressor gene in human cancer, plays important roles in proliferation, apoptosis and cell cycle in many type tumor cells. Recent studies have demonstrated that PI3K/AKT pathway is the key pathway by which PTEN displays antioncogenic functions . Our results showed that PTEN expression level was downregulated, and pAKT expression level was increased in TNBC cells after overexpression of miR-454. These results indicated miR-454 mediated proliferation of TNBC cells by targeting PI3K/AKT signaling pathway. Further studies are needed to demonstrate the molecular mechanism.
MiR-454 might act as an oncogene to regulate tumorigenesis and malignant progression of TNBC. Our study suggests miR-454 might be used as a potential biomarker to predict the radiotherapy response and prognosis in TNBC.
QL and JL performed the experiments, XM and RP analyzed the results, JL designed the project. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Availability of data and materials
Consent for publication
The study has been reviewed and approved by the Ethics Committee of Jilin University.
This project is supported by Young Scientist Award Jilin Province, China (201503102).
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- Du XL, Fox EE, Lai D. Competing causes of death for women with breast cancer and change over time from 1975 to 2003. Am J Clin Oncol. 2008;31:105–16.View ArticlePubMedPubMed CentralGoogle Scholar
- Gebski V, Lagleva M, Keech A, Simes J, Langlands AO. Survival effects of postmastectomy adjuvant radiation therapy using biologically equivalent doses: a clinical perspective. J Natl Cancer Inst. 2006;98:26–38.View ArticlePubMedGoogle Scholar
- Tornberg S, Kemetli L, Lynge E, Helene Olsen A, Hofvind S, Wang H, et al. Breast cancer incidence and mortality in the Nordic capitals, 1970–1998. Trends related to mammography screening programmes. Acta Oncol. 2006;45:528–35.View ArticlePubMedGoogle Scholar
- Anders C, Carey LA. Understanding and treating triple-negative breast cancer. Oncology. 2008;22:1233–9 (discussion 1239–40, 1243).PubMedPubMed CentralGoogle Scholar
- Lehmann BD, Jovanović B, Chen X, Estrada MV, Johnson KN, Shyr Y, et al. Refinement of triple-negative breast cancer molecular subtypes: implications for neoadjuvant chemotherapy selection. PLoS ONE. 2016;11:e0157368.View ArticlePubMedPubMed CentralGoogle Scholar
- Pogoda K, Niwińska A, Murawska M, Pieńkowski T. Analysis of pattern, time and risk factors influencing recurrence in triple-negative breast cancer patients. Med Oncol. 2013;30:388.View ArticlePubMedPubMed CentralGoogle Scholar
- Linklater ES, Tovar EA, Essenburg CJ, Turner L, Madaj Z, Winn ME, et al. Targeting MET and EGFR crosstalk signaling in triple-negative breast cancers. Oncotarget. 2016;7:69903–15.PubMedPubMed CentralGoogle Scholar
- Gray MJ, Gong J, Hatch MM, Nguyen V, Hughes CC, Hutchins JT, et al. Phosphatidylserine-targeting antibodies augment the anti-tumorigenic activity of anti-PD-1 therapy by enhancing immune activation and downregulating pro-oncogenic factors induced by T-cell checkpoint inhibition in murine triple-negative breast cancers. Breast Cancer Res. 2016;18:50.View ArticlePubMedPubMed CentralGoogle Scholar
- Krol J, Loedige I, Filipowicz W. The widespread regulation of microRNA biogenesis, function and decay. Nat Rev Genet. 2010;11:597–610.PubMedGoogle Scholar
- Zhang B, Pan X, Cobb GP, Anderson TA. microRNAs as oncogenes and tumor suppressors. Dev Biol. 2007;302:1–12.View ArticlePubMedGoogle Scholar
- Ma L, Teruya-Feldstein J, Weinberg RA. Tumour invasion and metastasis initiated by microRNA-10b in breast cancer. Nature. 2007;449:682–8.View ArticlePubMedGoogle Scholar
- Gabriely G, Teplyuk NM, Krichevsky AM. Context effect: microRNA-10b in cancer cell proliferation, spread and death. Autophagy. 2011;7:1384–6.View ArticlePubMedGoogle Scholar
- Huang TH, Wu F, Loeb GB, Hsu R, Heidersbach A, Brincat A, et al. Up-regulation of miR-21 by HER2/neu signaling promotes cell invasion. J Biol Chem. 2009;284:18515–24.View ArticlePubMedPubMed CentralGoogle Scholar
- Han M, Wang Y, Liu M, Bi X, Bao J, Zeng N, et al. MiR-21 regulates epithelial-mesenchymal transition phenotype and hypoxia-inducible factor-1α expression in third-sphere forming breast cancer stem cell-like cells. Cancer Sci. 2012;103:1058–64.View ArticlePubMedGoogle Scholar
- Zhang CM, Zhao J, Deng HY. MiR-155 promotes proliferation of human breast cancer MCF-7 cells through targeting tumor protein 53-induced nuclear protein 1. J Biomed Sci. 2013;20:79.View ArticlePubMedPubMed CentralGoogle Scholar
- Ferracin M, Bassi C, Pedriali M, Pagotto S, D’Abundo L, Zagatti B, et al. miR-125b targets erythropoietin and its receptor and their expression correlates with metastatic potential and ERBB2/HER2 expression. Mol Cancer. 2013;12:130.View ArticlePubMedPubMed CentralGoogle Scholar
- Volinia S, Galasso M, Sana ME, Wise TF, Palatini J, Huebner K, et al. Breast cancer signatures for invasiveness and prognosis defined by deep sequencing of microRNA. Proc Natl Acad Sci USA. 2012;109:3024–9.View ArticlePubMedPubMed CentralGoogle Scholar
- Sun L, Wang Q, Gao X, Shi D, Mi S, Han Q. MicroRNA-454 functions as an oncogene by regulating PTEN in uveal melanoma. FEBS Lett. 2015;589:2791–6.View ArticlePubMedGoogle Scholar
- Liu L, Nie J, Chen L, Dong G, Du X, Wu X, et al. The oncogenic role of microRNA-130a/301a/454 in human colorectal cancer via targeting Smad4 expression. PLoS ONE. 2013;8:e55532.View ArticlePubMedPubMed CentralGoogle Scholar
- Zhu DY, Li XN, Qi Y, Liu DL, Yang Y, Zhao J, et al. MiR-454 promotes the progression of human non-small cell lung cancer and directly targets PTEN. Biomed Pharmacother. 2016;81:79–85.View ArticlePubMedGoogle Scholar
- Yu L, Gong X, Sun L, Yao H, Lu B, Zhu L. miR-454 functions as an oncogene by inhibiting CHD5 in hepatocellular carcinoma. Oncotarget. 2015;6:39225–34.View ArticlePubMedPubMed CentralGoogle Scholar
- Liang HL, Hu AP, Li SL, Xie JP, Ma QZ, Liu JY. MiR-454 prompts cell proliferation of human colorectal cancer cells by repressing CYLD expression. Asian Pac J Cancer Prev. 2015;16:2397–402.View ArticlePubMedGoogle Scholar
- Cao ZG, Li JJ, Yao L, Huang YN, Liu YR, Hu X, et al. High expression of microRNA-454 is associated with poor prognosis in triple-negative breast cancer. Oncotarget. 2016;7:64900–9.PubMedPubMed CentralGoogle Scholar
- Zhang L, Zhou Z, Mei X, Yang Z, Ma J, Chen X, et al. Intraoperative radiotherapy versus whole-breast external beam radiotherapy in early-stage breast cancer: a systematic review and meta-analysis. Medicine. 2015;94:e1143.View ArticlePubMedPubMed CentralGoogle Scholar
- Abdulkarim BS, Cuartero J, Hanson J, Deschênes J, Lesniak D, Sabri S. Increased risk of locoregional recurrence for women with T1-2N0 triple-negative breast cancer treated with modified radical mastectomy without adjuvant radiation therapy compared with breast-conserving therapy. J Clin Oncol. 2011;29:2852–8.View ArticlePubMedPubMed CentralGoogle Scholar
- Dent R, Hanna WM, Trudeau M, Rawlinson E, Sun P, Narod SA. Pattern of metastatic spread in triple-negative breast cancer. Breast Cancer Res Treat. 2009;115:423–8.View ArticlePubMedGoogle Scholar
- López-Knowles E, O’Toole SA, McNeil CM, Millar EK, Qiu MR, Crea P, et al. PI3K pathway activation in breast cancer is associated with the basal-like phenotype and cancer-specific mortality. Int J Cancer. 2010;126:1121–31.View ArticlePubMedGoogle Scholar
- Cully M, You H, Levine AJ, Mak TW. Beyond PTEN mutations: the PI3K pathway as an integrator of multiple inputs during tumorigenesis. Nat Rev Cancer. 2006;6:184–92.View ArticlePubMedGoogle Scholar