Dysregulated miR-671-5p / CDR1-AS / CDR1 / VSNL1 axis is involved in glioblastoma multiforme

Abstract

MiR-671-5p is encoded by a gene localized at 7q36.1, a region amplified in human glioblastoma multiforme (GBM), the most malignant brain cancer. To investigate whether expression of miR-671-5p were altered in GBM, we analyzed biopsies from a cohort of forty-five GBM patients and from five GBM cell lines. Our data show significant overexpression of miR-671-5p in both biopsies and cell lines. By exploiting specific miRNA mimics and inhibitors, we demonstrated that miR-671-5p overexpression significantly increases migration and to a less extent proliferation rates of GBM cells. Through a combined in silico and in vitro approach, we identified CDR1-AS, CDR1, VSNL1 as downstream miR-671-5p targets in GBM. Expression of these genes significantly decreased both in GBM biopsies and cell lines and negatively correlated with that of miR-671-5p. Based on our data, we propose that the axis miR-671-5p / CDR1-AS / CDR1 / VSNL1 is functionally altered in GBM cells and is involved in the modification of their biopathological profile.

Keywords: cell networks; circular RNAs (circRNAs); glioblastoma multiforme (GBM); microRNAs (miRNAs); non coding RNAs (ncRNAs).

Figure 1. Expression of miR-671-5p, miR-671-3p, miR-21 and miR-7 in GBM biopsies

A. and cell lines B. Expression values are reported as box plots with whiskers from minimum to maximum to represent −1*ΔCt, both in GBM biopsies as in controls (A), and as mean of fold change (FC) ± Standard Deviation versus normal brain (B). miR-99a and miR-92a were used as reference genes in experiments on biopsies and cell lines, respectively. *p-value < 0.05, **p-value < 0.01, ***p-value < 0.001, Student's t-test (n = 3).

Figure 2. CDR1-AS, CDR1, VSNL1 and CHPF2 expression in GBM biopsies

A. and cell lines B. Expression values are reported as box plots with whiskers from minimum to maximum to represent −1*ΔCt, both in GBM biopsies and controls (A), and as mean of fold change (FC) ± Standard Deviation versus normal brain (B). Western blot of CHPF2 and VSNL1 in GBM cell lines and normal brain tissue C. *p-value < 0.05, **p-value < 0.01, ***p-value < 0.001, Student's t-test (n = 3).

Figure 3. Scatter plots showing correlation between expression of miR-671-5p and its targets

Spearman's nonparametric rank correlation coefficients were calculated using ΔCt values of miR-671-5p and its targets CDR1-AS A. CDR1 B. VSNL1 C. CHPF2 D. and ΔCt values of CDR1-AS and CDR1 E. See text for details.

Figure 4. Expression of CDR1-AS, CDR1, VSNL1 in DBTRG, SNB-19, U-87 MG cell lines after transfection with miR-671-5p mimics (miR-671-5p Mim) or inhibitors (miR-671-5p Inh)

Expression values are reported as mean of fold change (FC) ± Standard Deviation versus scramble molecules (NC) - transfected cells. *p-value < 0.05; **p-value < 0.01, Student's t-test (n = 3).

Figure 5. Involvement of miR-671-5p in DBTRG, SNB-19, U-87 MG migration

MiR-671-5p mimics stimulate cell migration with respect to scramble molecules (NC Mim) - transfected DBTRG A. SNB-19 B. U-87 MG C. cells in a wound-healing assay. Data are represented as mean percentage ± Standard Deviation of open wound area of three independent biological replicates, 24 h after scratch. Magnification: × 8. Results are representative of at least three randomly selected areas, assayed for each well. *p-value < 0.05; **p-value < 0.01, Student's t-test.

Figure 6. MTT assay in GBM cell lines DBTRG

(A), SNB-19 (B), U-87 MG (C). Data are reported as percentages of viable cells, relative to controls. Experiments were performed as six biological replicates. *P < 0.05, **P < 0.01, Student's t-test. Ctrl (not transfected cells); NC Mim (Cells transfected with scramble molecules of miRNA mimics); 671–5p Mim (Cells transfected with miR-671-5p mimics); NC Inh (Cells transfected with scramble molecules of miRNA inhibitors); 671–5p Inh (Cells transfected with miR-671-5p inhibitors).

Figure 7. Network of CDR1 and VSNL1 (candidate nodes) plus their first neighbor interactants

See legend within the figure and text for further details.