miR-3188 regulates nasopharyngeal carcinoma proliferation and chemosensitivity through aFOXO1-modulated positive feedback loop with mTOR–p-PI3K/AKT-c-JUN
research target：miR-3188
Periodicals：nature communications
IF：11.47
Cooperative Unit：Southern Medical University
Time of publication：April，2016
Summary
The biological role of miR-3188 has not yet been reported in the context of cancer. In this study, we observe that miR-3188 not only reduces cell-cycle transition and proliferation, but also significantly prolongs the survival time of tumour-bearing mice as well as sensitizes cells to 5-FU. Mechanistic analyses indicate that miR-3188 directly targets mTOR to inactivate p-PI3K/p-AKT/c-JUN and induces its own expression. This feedback loop further suppresses cell-cycle signalling through the p-PI3K/p-AKT/p-mTOR pathway. Interestingly, we also observe that miR-3188 direct targeting of mTOR is mediated by FOXO1 suppression of p-PI3K/p-AKT/c-JUN signalling. In clinical samples, reduced miR-3188 is an unfavourable factor and negatively correlates with mTOR and c-JUN levels but positively correlates with FOXO1 expression. Our studies demonstrate that as a tumour suppressor, miR-3188 directly targets mTOR to stimulate its own expression and participates in FOXO1-mediated repression of cell growth, tumorigenesis and NPC chemotherapy resistance.
Research Background
MicroRNAs (miRNAs or miRs) play important roles in development, cellular differentiation, proliferation, cell-cycle control and cell death, and have been implicated in a variety of human diseases, including cancer. A growing body of evidence has demonstrated the importance of miRNAs in managing chemotherapy efficacy in multiple human cancers. Despite being one of the original miRNAs discovered, the biological role of miR-3188 and its molecular mechanisms underlying cancer initiation and progression have not been reported.
Nasopharyngeal carcinoma (NPC) is a tumour type arising from the epithelial cells that line the nasopharynx. It is common in certain regions of East Asia and Africa, with Epstein-Barr virus (EBV) exposure, diet and genetic factors implicated in its aetiology. In recent studies, abnormal expression of miRNAs was been broadly implicated in the pathogenesis of NPC. For example, EBV-encoded miRNA BART1 induces tumour metastasis by regulating the PTEN-dependent pathways. In addition, tumour suppressor PDCD4 modulates miR-184-mediated direct suppression of c-MYC and BCL2-blocking cell growth and survival.
FOXO1 is a transcription factor and a member of the FOXO subfamily of the Forkhead/winged helix family. The phosphorylation of FOXO1 by AKT leads to its inactivation after nuclear to cytoplasmic translocation. Recent evidence suggested that LMP1 silencing slows cell growth and enhances chemosensitivity through inhibition of the AKT signalling pathway and its downstream factor phospho-FOXO1 in EBV-positive NPC cell line. Elevated levels of phosphorylated AKT also correlated with phospho-FOXO1 in NPC samples. However, the detailed role of FOXO1 in the suppression of NPC cell growth remains unclear.
This study found that miR-3188 targets mTOR and FOXO1 in nasopharyngeal carcinoma cells, and found that FOXO1 miR-3188-mTOR–p-PI3K/AKT-c-JUN signaling pathway, which is regulated by FOXO1. Activation of this pathway inhibits tumor proliferation and increases its sensitivity to 5-fluorouracil.
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EMSA and ChIP-PCR experiments jointly confirmed the c-JUN transcription factor binding site on miR-3188:
(c) EMSA result was shown from nuclear proteins extracted from SUNE1 and HONE1-EBV cells after incubation with individual DIG-ddUTP-labelled oligonucleotide probes (lanes 2–6, 8–12). The free probe of labelled c-JUN was run in lanes 1 and 7 as a control. A 100-fold excess of unlabelled c-JUN-WT was used to compete with c-JUN binding (lanes 6 and 12, compared with lanes 2 and 8). A 100-fold excess of unlabelled mutated c-JUN-A, c-JUN-B and c-JUN-C was used to compete with binding of respective labelled probes (lanes 3–5 and lanes 9–11 compared with lanes 2 and 8).
(d) PCR gel showing amplification of c-JUN-binding sites A, B and C after ChIP using antibody against c-JUN. The gel figures were accompanied by the locations of molecular weight markers.
In situ hybridization experiments confirmed the expression of miR-3188 in NPC and NP samples:
(a) Weak expression of miR-3188 in NP samples;
(b) strong staining of miR-3188 in NP samples;
(c) negative expression of miR-3188 in NPC samples;
(d) strong staining of miR-3188 in NPC samples (original magnification × 400). Scale bar: 30 μm.
References
Zhao M, Luo R, Liu Y, et al. miR-3188 regulates nasopharyngeal carcinoma proliferation and chemosensitivity through a FOXO1-modulated positive feedback loop with mTOR-p-PI3K/AKT-c-JUN[J]. Nature Communications, 2016, 7.
