MicroRNA-16-1-3p Represses Breast Tumor Growth and Metastasis by Inhibiting PGK1-Mediated Warburg Effect

Abstract

The Warburg effect (aerobic glycolysis) is a hallmark of cancer and is becoming a promising target for diagnosis and therapy. Phosphoglycerate kinase 1 (PGK1) is the first adenosine triphosphate (ATP)-generating glycolytic enzyme in the aerobic glycolysis pathway and plays an important role in cancer development and progression. However, how microRNAs (miRNAs) regulate PGK1-mediated aerobic glycolysis remains unknown. Here, we show that miR-16-1-3p inhibits PGK1 expression by directly targeting its 3'-untranslated region. Through inhibition of PGK1, miR-16-1-3p suppressed aerobic glycolysis by decreasing glucose uptake, lactate and ATP production, and extracellular acidification rate, and increasing oxygen consumption rate in breast cancer cells. Aerobic glycolysis regulated by the miR-16-1-3p/PGK1 axis is critical for modulating breast cancer cell proliferation, migration, invasion and metastasis in vitro and in vivo. In breast cancer patients, miR-16-1-3p expression is negatively correlated with PGK1 expression and breast cancer lung metastasis. Our findings provide clues regarding the role of miR-16-1-3p as a tumor suppressor in breast cancer through PGK1 suppression. Targeting PGK1 through miR-16-1-3p could be a promising strategy for breast cancer therapy.

Keywords: PGK1; cell proliferation; metastasis; miR-16-1-3p; the Warburg effect.

FIGURE 1

miR-16-1-3p suppresses PGK1 expression by directly targeting its 3′-UTR in breast cancer cells. (A) HEK293T cells were transfected with negative control (NC) for miRNAs or mimics of candidate miRNAs as indicated. The representative immunoblot shows PGK1 expression. α-Tubulin was used as a loading control. (B,C) Immunoblot analysis of PGK1 expression in ZR75-1 and MDA-MB-231 breast cancer cells transfected with (B) NC or miR-16-1-3p mimics, or (C) scramble for miRNA inhibitors or miR-16-1-3p inhibitor (anti-miR-16-1-3p). Right histograms indicate relative miR-16-1-3p expression by RT-qPCR. (D) RT-qPCR analysis of PGK1 mRNA expression in ZR75-1 and MDA-MB-231 cells transfected as in panels (B,C). (E) miRNA luciferase reporter assays of ZR75-1 and MDA-MB-231 cells transfected with wild-type (WT) or mutated (MUT) PGK1 reporter plus miR-16-1-3p mimics. The top panel indicates WT and MUT forms of putative miR-16-1-3p target sequences of PGK1 3′-UTR. Red font indicates the putative miR-16-1-3p binding sites within human PGK1 3′-UTR. Blue font shows the mutations introduced into the PGK1 3′-UTR. Data shown are mean ± SD of triplicate measurements that were repeated three times with similar results. **P < 0.01.

FIGURE 2

The miR-16-1-3p/PGK1 axis modulates aerobic glycolysis in breast cancer cells. (A) Glucose uptake and the production of lactate and ATP were examined in MDA-MB-231 cells transfected with miR-16-1-3p or miR-16-1-3p plus PGK1 expression vector as indicated. EV, empty vector. (B) Glucose uptake and the production of lactate and ATP were examined in MDA-MB-231 cells transfected with anti-miR-16-1-3p, PGK1 siRNA or anti-miR-16-1-3p plus PGK1 siRNA. Ctrl siRNA, control siRNA. Representative immunoblot shows PGK1 expression, and RT-qPCR analysis indicates miR-16-1-3p expression (A,B). (C) ECAR and OCR assays of MDA-MB-231 cells transfected as in panel (A). (D) ECAR and OCR assays of MDA-MB-231 cells transfected as in panel (B). Data shown are mean ± SD of quintuplicate measurements that were repeated three times with similar results [panels (A,B) for glucose uptake and the production of lactate and ATP]. Data shown are mean ± SD of triplicate measurements that were repeated three times with similar results (A and B for RT-qPCR analysis). **P < 0.01 (A,B). Data shown are mean ± SD of quintuplicate measurements that were repeated 3 times with similar results (C,D). **P < 0.01 vs NC plus EV or Scramble plus Ctrl shRNA (C,D).

FIGURE 3

Aerobic glycolysis is responsible for miR-16-1-3p modulation of breast cancer cell proliferation. The proliferation curve shows ZR75-1 (A) and MDA-MB-231 (B) cells transfected with anti-miR-16-1-3p or scramble and treated with 2.5 mM 2-DG as indicated. RT-qPCR reveals miR-16-1-3p expression. Data shown are mean ± SD of triplicate measurements that were repeated three times with similar results. **P < 0.01.

FIGURE 4

miR-16-1-3p inhibits proliferation, migration and invasion by inhibiting PGK1 expression in breast cancer cells. (A) The proliferation curve of MDA-MB-231 cells transfected with miR-16-1-3p mimics or miR-16-1-3p mimics plus PGK1 expression plasmid as indicated. Immunoblot analysis shows PGK1 expression. RT-qPCR indicates miR-16-1-3p expression. (B,C) Wound healing (B) and invasion (C) assays of MDA-MB-231 cells transfected as in panel (A). Histograms denote relative cell migration (B) and invasion (C). (D) The proliferation curve of MDA-MB-231 cells transfected with anti-miR-16-1-3p, PGK1 siRNA or anti-miR-16-1-3p plus PGK1 siRNA as indicated. Immunoblot analysis shows PGK1 expression. RT-qPCR indicates miR-16-1-3p expression. (E,F) Wound healing (E) and invasion (F) assays of MDA-MB-231 cells transfected as in panel (D). Histograms show relative cell migration (E) and invasion (F). All values shown are mean ± SD of triplicate measurements that were repeated three times with similar results. (G) Immunoblot analysis of E-Cadherin and Vimentin expression in MDA-MB-231 cells transfected with NC or miR-16-1-3p mimics. (H) Immunoblot analysis of E-Cadherin and Vimentin expression in MDA-MB-231 cells transfected with scramble or miR-16-1-3p inhibitor. **P < 0.01.

FIGURE 5

The miR-16-1-3p/PGK1 axis regulates breast tumor growth and metastasis in nude mice. (A) MDA-MB-231 cells stably infected with lentivirus harboring PGK1 shRNA or control shRNA (Ctrl shRNA) were treated with antagomiR-16-1-3p or antagomiR-NC and injected into nude mice as indicated. After 42 days, mice were euthanized to harvest tumors. Images of all xenograft tumors excised at day 42 are shown. The tumor growth curves were plotted. Lactate production of representative tumor tissues was measured. miR-16-1-3p and PGK1 expression of representative tumor tissues was determined by RT-qPCR and immunoblot, respectively. Tumor volumes are presented as means ± SD (n = 8). **P < 0.01 at day 42. Data shown are mean ± SD of quintuplicate measurements for lactate production that were repeated 3 times with similar results. **P < 0.01. (B) Representative bioluminescence images at 30 days of nude mice injected by tail vein with MDA-MB-231 cells expressing firefly luciferase and the indicated constructs (n = 6). The luminescence signal is represented by an overlaid false-color image with the signal intensity indicated by the scale (right panel). (C) Representative H&E-stained sections of the lung tissues from (B). The number of tumor nodules are shown (right panel). **P < 0.01 (B,C).

FIGURE 6

Correlation of miR-16-1-3p expression with PGK1 expression and metastasis in breast cancer patients. (A) Correlation between miR-16-1-3p and PGK1 expression in 91 breast cancer patients. miR-16-1-3p and PGK1 expression was assessed by MISH and IHC, respectively. Scale bar: 100 μm. The P value was generated using Spearman’s Rank Correlation test. (B) Association of miR-16-1-3p expression with metastasis. The P value was generated using independent t test.