Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2020 Aug 5:10:1371.
doi: 10.3389/fonc.2020.01371. eCollection 2020.

MicroRNA-27b-3p Promotes Tumor Progression and Metastasis by Inhibiting Peroxisome Proliferator-Activated Receptor Gamma in Triple-Negative Breast Cancer

Song-Jie Shen  1 , Yu Song  1 , Xin-Yu Ren  2 , Ya-Li Xu  1 , Yi-Dong Zhou  1 , Zhi-Yong Liang  2 , Qiang Sun  1
Affiliations

MicroRNA-27b-3p Promotes Tumor Progression and Metastasis by Inhibiting Peroxisome Proliferator-Activated Receptor Gamma in Triple-Negative Breast Cancer

Song-Jie Shen et al. Front Oncol. .

Abstract

Introduction: The role and underlying mechanisms of miR-27b-3p in triple-negative breast cancer (TNBC) remains unclear. Methods: miR-27b-3p expression level was evaluated in 99 TNBC patients with a median follow-up time of 133 months. The biological functions of miR-27b-3p by targeting PPARG were assessed by luciferase reporter assay, CCK-8 assay, Transwell assay, wound healing assay, western blot analysis and xenograft models. Results: High level of miR-27b-3p expression was found to confer poor prognosis in TNBC patients. MiR-27b-3p overexpression increased TNBC cell proliferation, migration, invasion, and metastasis. Our data suggested peroxisome proliferator-activated receptor gamma (PPARG) was a target of miR-27b-3p. The capacity of miR-27b-3p to induce TNBC progression and metastasis depended on its inhibition of the PPARG expression. Furthermore, restoring PPARG expression reversed the effect of miR-27b-3p overexpression. Mechanistically, miR-27b-3p regulated metastasis-related pathways through PPARG by promoting epithelial-mesenchymal transition. By suppressing PPARG, miR-27b-3p could also activate transcription factors Snail and NF-κB, thereby promoting metastasis. Conclusions: miR-27b-3p promotes TNBC progression and metastasis by inhibiting PPARG. MiR-27b-3p may be a potential prognostic marker of TNBC, and PPARG may be a potential molecular therapeutic target of TNBC.

Keywords: PPARG; metastasis; microRNA-27b-3p; prognosis; triple-negative breast cancer.

PubMed Disclaimer

Figures

Figure 1
Figure 1
High miR-27b-3p expression indicated worse prognosis in TNBC patients. Kaplan-Meier curves showed the disease-free survival (A) and overall survival (B) of TNBC patients with high or low expression of miR-27b-3p. P-values were computed by a log-rank test.
Figure 2
Figure 2
MiR-27b-3p promotes cell proliferation, migration, and invasion in TNBC. MDA-MB-231 cells were transiently transfected with miR-27b-3p mimics or inhibitors. (A) The relative miR-27b-3p levels were detected by RT-PCR in MDA-MB-231 cells transfected with blank, negative control (NC), miR-27b-3p inhibitors or miR-27b-3p mimics. (B) CCK-8 proliferation test showed that overexpression of miR-27b-3p promoted MDA-MB-231 cells proliferation and miR-27b-3p inhibitors decreased cell proliferation. (C,D) Transwell migration assay. Overexpression of miR-27b-3p increased cell migration significantly, while inhibition of miR-27b-3p decreased cell migration. (E,F) Transwell invasion assay. Similar to the cell migration experiments, a significantly higher invasion capability was detected in the miR-27b-3p mimics group. MDA-MB-231 cells transfected with miR-27b-3p inhibitors showed significant inhibition of cell invasion. (G,H) Wound healing assay. The wound healing rates were compared among MDA-MB-231 cells transfected with blank, NC, miR-27b-3p inhibitors, and miR-27b-3p mimics at 24 h. For (A,B,D,F,H), results are shown as mean ± SD of 3 times of experiments in each group. **P < 0.01, ***P < 0.001; NC, negative control of transfection vectors.
Figure 3
Figure 3
PPARG is the target of miR-27b-3p in TNBC cells. (A) The binding sequence of miR-27b-3p and 3'-UTR of PPARG. The mutant sequence of 3'-UTR of PPARG was generated in the complementary site for the seed region of miR-27b-3p (WT, wild type; MUT, mutant type). (B) Luciferase reporter assay confirmed that miR-27b-3p repressed the luciferase activity of wild-type PPARG-3'UTR, while the repression effect was not detected with mutant PPARG-3'UTR. (C) RT-PCR analysis showed the mRNA level of PPARG could be up-regulated by miR-27b-3p inhibitors and down-regulated by miR-27b-3p mimics (ACTB used as a loading control). (D,E) Western blot analysis showed that miR-27b-3p mimics inhibited the protein expression level of PPARG, while miR-27b-3p inhibitors increased the expression of PPARG (GAPDH served as a loading control). For (B,C,E), results are shown as mean ± SD of 3 times of experiments in each group. *P < 0.05, **P < 0.01, ***P < 0.001; NC, negative control of transfection vectors.
Figure 4
Figure 4
Effect of miR-27b-3p on cell proliferation and invasion depends on its inhibitory effect on PPARG expression in TNBC cells. The MDA-MB-231 cells were transfected with miR-27b-3p-3p NC, miR-27b-3p-3p mimics, or miR-27b-3p-3p mimics and treated with PPARG agonist (rosiglitazone, 80 μmol/L) for 24 h. (A) CCK-8 test assay showed that overexpression of miR-27b-3p could promote cell proliferation. However, this promotion effect of miR-27b-3p could be reversed by PPARG agonist. (B,C) Wound healing assay showed that PPARG agonist abolished the effect of miR-27b-3p mimics on the wound-healing rate of MDA-MB-231 cells. (D,E) Transwell invasion assay. The number of invasive cells in the miR-27b-3p mimics group were significantly higher than those of the control group, which were significantly decreased by PPARG agonist. For (A,C,E), results are shown as mean ± SD of 3 times of experiments in each group. ***P < 0.001; NC, negative control of transfection vectors.
Figure 5
Figure 5
MiR-27b-3p promotes tumor growth in immunodeficient murine models. The MDA-MB-231 cells were transfected with miR-27b-3p NC, miR-27b-3p mimics, or miR-27b-3p mimics and treated with PPARG agonist (rosiglitazone, 80 μmol/L) for 24 h. Then, these cells were injected subcutaneously into the mammary fat pad of female nude BALB/c mice (three mice in each group). (A,B) Tumor xenografts in the miR-27b-3p mimics group significantly overweighed those in the negative control group, but the advantage was canceled by PPARG agonist. (C) Primary mammary tumor volume in mice injected with miR-27b-3p NC, miR-27b-3p mimics, or miR-27b-3p mimics with PPARG agonist MDA-MB-231 cells. For (B,C), results are shown as mean ± SD of 3 times of experiments in each group. *P < 0.05, **P < 0.01; NC, negative control of transfection vectors.
Figure 6
Figure 6
MiR-27b-3p targets PPARG to regulate the EMT process and metastasis-related pathways. (A) Western blot analysis of PPARG, E-cadherin, Vimentin, Snail, and NF-κB after MDA-MB-231 cells were transfected by miR-27b-3p mimics or miR-27b-3p mimics together with PPARG agonist (GAPDH served as a loading control). (B) Relative expression analysis showed miR-27b-3p mimics activated EMT process and activated the expression of the metastasis-related transcription factors, while these effects were significantly repressed by PPARG agonists. For (B), results are shown as mean ± SD of 3 times of experiments in each group. *P < 0.05, **P < 0.01, ***P < 0.001; NC, negative control of transfection vectors.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. (2018) 68:394–424. 10.3322/caac.21492 - DOI - PubMed
    1. Garrido-Castro AC, Lin NU, Polyak K. Insights into molecular classifications of triple-negative breast cancer: improving patient selection for treatment. Cancer Discov. (2019) 9:176–98. 10.1158/2159-8290.CD-18-1177 - DOI - PMC - PubMed
    1. Bianchini G, Balko JM, Mayer IA, Sanders ME, Gianni L. Triple-negative breast cancer: challenges and opportunities of a heterogeneous disease. Nat Rev Clin Oncol. (2016) 13:674–90. 10.1038/nrclinonc.2016.66 - DOI - PMC - PubMed
    1. Denkert C, Liedtke C, Tutt A, von Minckwitz G. Molecular alterations in triple-negative breast cancer-the road to new treatment strategies. Lancet. (2017) 389:2430–42. 10.1016/S0140-6736(16)32454-0 - DOI - PubMed
    1. da Silva JL, Cardoso Nunes NC, Izetti P, de Mesquita GG, de Melo AC. Triple negative breast cancer: a thorough review of biomarkers. Crit Rev Oncol Hematol. (2020) 145:102855. 10.1016/j.critrevonc.2019.102855 - DOI - PubMed