MicroRNA-608 and MicroRNA-34a Regulate Chordoma Malignancy by Targeting EGFR, Bcl-xL and MET

<div><p>Chordomas are rare malignant tumors that originate from the notochord remnants and occur in the skull base, spine and sacrum. Due to a very limited understanding of the molecular pathogenesis of chordoma, there are no adjuvant and molecular therapies besides surgical resection and radiation therapy. microRNAs (miRNAs) are small noncoding regulatory RNA molecules with critical roles in cancer. The role of miRNAs in chordomas is mostly unknown. We uncover microRNA-608 (miR-608) and microRNA-34a (miR-34a) as novel tumor suppressive microRNAs that regulate malignancy in chordoma. We find that miR-608 and miR-34a expressions are downregulated in human chordoma cell lines and primary cells at least partially via alteration of their genes’ copy numbers. We identify the commonly deregulated oncogenes EGFR and Bcl-xL as direct targets of miR-608 and the receptor tyrosine kinase MET as direct target of miR-34a. We show that EGFR and MET activations promote chordoma cell proliferation and invasion and that pharmacological inhibition of EGFR and MET inhibits chordoma cell proliferation and survival. We demonstrate that restoration of miR-608 and miR-34a inhibits cell proliferation and invasion and induces apoptosis in chordoma cells. We find that miR-34a inversely correlates with MET expression and miR-608 inversely correlates with EGFR expression in chordoma cells. These findings demonstrate for the first time that miR-608 and miR-34a regulate chordoma malignancy by regulating EGFR, MET and Bcl-xL.</p></div

miR-608 and miR-34a are downregulated via gene copy number alteration, and miR-608 and miR-34a expressions inversely correlate with EGFR and MET in chordoma cells.

<p>A) miR-608 and miR-34a levels in chordoma cell lines and primary cells were measured by qRT-PCR and compared with those in fibroblasts, B) lystaes from chordoma cells were immunoblotted for EGFR and normalized to β-actin (left panel), and the level of miR-608 was correlated with EGFR protein level (right panel) (R² = 0.8, P<0.05). C) MET protein levels in chordoma cells were determined by immunoblotting (left panel) and miR-34a levels correlated with MET protein (right panel) (R² = 0.0.61, P<0.05). D) miR-608 gene copy numbers in chordoma cells were determined by q-PCR (left panel) and miR-608 expression levels were correlated with gene copy number (R² = 0.77, P<0.05) (right panel). E) miR-34a gene copy numbers in chordoma cells were determined by q-PCR (left panel) and miR-34a expression levels are correlated with gene copy number (R² = 0.51, P<0.05).</p

EGFR and MET expression and gene amplification in chordoma in published immunohistochemical studies.

<p>EGFR and MET expression and gene amplification in chordoma in published immunohistochemical studies.</p

miR-608 downregulates EGFR and Bcl-xL by directly binding to their mRNA 3′UTR and miR-34a downregulates MET by directly binding MET 3′UTR.

<p>A) Predicted binding sequences of miR-608 in the 3′UTR sequences of EGFR and Bcl-xL mRNA; B) Predicted binding sequences of miR-34a in the 3′UTR sequence of MET mRNA; C), D) UCH1 and C24 cells were transfected with pre-miR-608 (C) or pre-miR-34a (D) or control pre-miR for 48 hrs. Cell lysates were immunoblotted for EGFR or Bcl-xL (C) or MET (D), The results show that the miRNAs significantly inhibited these predicted target proteins in chordoma cells; E), F) UCH1 cells were transfected with pre-miR-608, pre-miR-34a or pre-miR-con and then with either EGFR 3′UTR, Bcl-xL 3′UTR, MET 3′UTR or control reporter plasmids together with β-Galactosidase (β-Gal) plasmid, and 3′UTR reporter activity was measured by a luciferase assay and normalized to β-Gal activity. The results show that miR-608 expression down-regulates EGFR and Bcl-xL luciferase activities (E) and that miR-34a expression repressed MET luciferase activity (F) in UCH1 cells. (* P<0.05)</p

microRNAs are differentially expressed in chordoma cells.

<p>Small RNAs were extracted from chordoma UCH1 and UCH2 cells and control fibroblasts. miRNA levels were measured using qRT-PCR relative to control U6B snRNA.</p