Identification of key microRNAs regulating ELOVL6 and glioblastoma tumorigenesis

Abstract

ELOVL fatty acid elongase 6 (ELOVL6) controls cellular fatty acid (FA) composition by catalyzing the elongation of palmitate (C16:0) to stearate (C18:0) and palmitoleate (C16:1n-7) to vaccinate (C18:1n-7). Although the transcriptional regulation of ELOVL6 has been well studied, the post-transcriptional regulation of ELOVL6 is not fully understood. Therefore, this study aims to evaluate the role of microRNAs (miRNAs) in regulating human ELOVL6. Bioinformatic analysis identified five putative miRNAs: miR-135b-5p, miR-135a-5p, miR-125a-5p, miR-125b-5p, and miR-22-3p, which potentially bind ELOVL6 3'-untranslated region (UTR). Results from dual-luciferase assays revealed that these miRNAs downregulate ELOVL6 by directly interacting with the 3'-UTR of ELOVL6 mRNA. Moreover, miR-135b-5p and miR-135a-5p suppress cell proliferation and migration in glioblastoma multiforme cells by inhibiting ELOVL6 at the mRNA and protein levels. Taken together, our results provide novel regulatory mechanisms for ELOVL6 at the post-transcriptional level and identify potential candidates for the treatment of patients with glioblastoma multiforme.

Keywords: Cell proliferation; Fatty acid; Glioblastoma; MicroRNA; Migration.

Fig. 1

Identification ofELOVL6-targeting miRNAs. A: Schematic representation of the ELOVL6 3′-UTR and the conserved miRNA target sites predicted by TargetScan. F1: Fragment 1, F2: Fragment 2, F3: Fragment 3. B–D: Luciferase activity of the dual-luciferase reporter vector with F1 (B), F2 (C), or F3 (D) of ELOVL6 3′-UTR cotransfected with empty vector (EV) or the predicted miRNA expressing vector in HEK293 cells. E: qRT-PCR analysis of ELOVL6 mRNA in HEK293 cells transfected with EV or the predicted miRNA expressing vector. F: HA-tagged ELOVL6 with the F1 of ELOVL6 3′-UTR was cotransfected with EV, miR-135b-5p, miR-135a-5p, miR-125a-5p, miR-125b-5p, or miR-22–3p into HEK293 cells, and protein levels of HA-ELOVL6 were analyzed by western blotting. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 compared with the EV control. 3′-UTR: 3′-untranslated region.

Fig. 2

Mutations in miRNA binding sites in the 3′-UTR ofELOVL6disturb the effects ofELOVL6-targeting miRNAs (A–C) Sequence alignments of binding sites of miR-135b-5p and miR-135a-5p (A), miR-125a-5p and miR 125b-5p (B), and miR-22–3p (C) in ELOVL6 3′-UTR from different species and mutations that were introduced into specific binding sites. The seed regions are highlighted. (D–F) Validation of specific target recognition using the dual-luciferase assay in miR-135b-5p and miR-135a-5p (D), miR-125a-5p and miR 125b-5p (E), and miR-22–3p (F). WT: Wild-type, Mut: Mutant. ns: not significant; *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 3

miR-135b-5p and miR-135a-5p decrease the abundance of ELOVL6 at both mRNA and protein levels in GBM cells (A) Alignment between mature miR-135b-5p and miR-135a-5p indicating one nucleotide difference. (B, C) Luciferase activity of the dual-luciferase reporter vector with the F1 of ELOVL6 3′-UTR cotransfected with miRNA mimics or negative control (NC) in U87-MG (B) and U118-MG cells (C). 3′-UTR WT containing wild-type ELOVL6 3′-UTR; 3′-UTR Mut containing mutations in specific miRNA target sites of ELOVL6 3′-UTR indicated in Fig. 2A; EV or predicted miRNA expressing vector in HEK293 cells. (D, E) Expression levels of ELOVL6 in U87-MG (D) and U118-MG cells (E) transfected with the miR-135b-5p mimic, miR-135a-5p mimic, or NC were measured by qRT-PCR. (F) HA-tagged ELOVL6 and F1 of ELOVL6 3′-UTR were cotransfected with the miR-135b-5p mimic, miR-135a-5p mimic, or NC into U87-MG and U118-MG cells, and levels of HA-ELOVL6 were analyzed by western blotting. (G, H) The palmitoyl-CoA elongation activity assay in U87-MG (G) and U118-MG GBM (H) cells transfected with the miR-135b-5p mimic, miR-135a-5p mimic, or NC. **P < 0.01; ***P < 0.001, ****P < 0.0001. GBM: glioblastoma multiforme.

Fig. 4

miR-135b-5p and miR-135a-5p regulate fatty acid composition in GBM cells (A) Fatty acid composition of U87-MG cells transfected with miR-135b-5p mimic, miR-135a-5p mimic or negative control (NC) mimic for 48 h (n = 4 per group). (B) The ratio of stearate (C18:0) to palmitate (C16:0) in U87-MG cells transfected with miR-135b-5p mimic, miR-135a-5p mimic or negative control mimic for 48 h (n = 4 per group). (C) Fatty acid composition of U118-MG cells transfected with miR-135b-5p mimic, miR-135a-5p mimic, or negative control mimic for 48 h (n = 4 per group). (D) The ratio of stearate (C18:0) to palmitate (C16:0) in U118-MG cells transfected with miR-135b-5p mimic, miR-135a-5p mimic, or negative control (NC) mimic for 48 h (n = 4 per group). *P < 0.05; **P < 0.01; and ***P < 0.001 vs. NC. N.D.: not detected.

Fig. 5

miR-135b-5p and miR-135a-5p suppress cell proliferation in glioblastoma cells by inhibiting ELOVL6 (A, B) Representative fluorescence microscopy images of EdU cell proliferation assay in U87-MG (A) and U118-MG (B) cells transfected with the miR-135b-5p mimic, miR-135a-5p mimic, or NC and ELOVL6 or EV. (C, D) Percentage of EdU-positive cells in U87-MG (C) and U118-MG (D) cells treated as in (A) and (B), respectively. ****P < 0.0001. EdU, 5-ethynyl-2′-deoxyuridine; DAPI, 4′,6-diamidino-2-phenylindole. EV: pcDNA 3.1 (+) empty vector, OE: overexpression.

Fig. 6

miR-135b-5p and miR-135a-5p suppress cell migration in glioblastoma cells by inhibiting ELOVL6 (A, B) Representative images of the wound healing assay in U87-MG (A) and U118-MG (B) cells transfected with the miR-135b-5p mimic, miR-135a-5p mimic, or NC and ELOVL6 or EV at 0, 12, and 24 h. (C, D) Percentage of the migrated area following the wound healing assay in U87-MG (C) and U118-MG (D) cells treated as in (A) and (B), respectively. **P < 0.01, ***P < 0.001 compared to NC. OE: overexpression, EV: pcDNA 3.1 (+) empty vector.