miR-330-5p suppresses glioblastoma cell proliferation and invasiveness through targeting ITGA5

Abstract

The present study intended to investigate the biological effects of miR-330-5p on glioblastoma (GBM) cell proliferation and invasiveness by targeting integrin α5 (ITGA5). The expressions of miR-330-5p and ITGA5 mRNA in GBM cell lines (U87, U251, and U373) and normal brain glial cell line (HEB) were detected using RT-qPCR. Protein expression of ITGA5 was examined using Western blot. The present study used MTT assay, colony formation assay, Transwell assay, wound healing assay, and flow cytometry analysis in order to determine the biological functions of GBM cells (including cell proliferation, invasion, migration, apoptosis, and cell cycle). The present study applied dual-luciferase reporter gene assay to identify the target relationship between miR-330-5p and ITGA5. miR-330-5p was low-expressed in GBM cell lines while ITGA5 was high-expressed compared with HEB. miR-330-5p could directly target ITGA5 as well as suppress its expression in GBM cells. Up-regulation of miR-330-5p and down-regulation of ITGA5 both have an inhibitory effect on cell proliferation, invasion, and migration. Meanwhile, they could also promote GBM cell apoptosis. miR-330-5p could suppress proliferation and invasion of GBM cells through targeting ITGA5.

Keywords: ITGA5; glioblastoma (GBM); invasiveness; miR-330-5p; proliferation.

Figure 1. The expressions of miR-330-5p and ITGA5 in GBM cells

(A) The expressions of miR-330-5p in GBM cell lines (U87, U251, and U373) were significantly lower than that in normal cell line (HEB) detected by qRT-PCR. (B) The mRNA expressions of ITGA5 in GBM cell lines were obviously higher than that in HEB cells detected by qRT-PCR. (C) The protein expression levels of ITGA5 were higher in GBM cell lines than in HEB cells, with GAPDH as internal control. *P<0.05 compared with HEB group.

Figure 2. miR-330-5p directly targeted ITGA5

(A) The binding site between mutant or wild-type ITGA5 3′-UTR and miR-330-5p was predicted by TargetScan. (B) Luciferase activity in U251 cells co-transfected with miR-330-5p mimics and wild-type ITGA5 3′-UTR was significantly lower than that of NC in ITGA5-wt group. *P<0.05 compared with NC group. (C) RT-qPCR results showed that cells transfected with miR-330-5p or siRNA-ITGA5 presented lower ITGA5 mRNA expression. (D) Expression levels of ITGA5 protein detected by Western blot suggested that miR-330-5p could suppress the expression of ITGA5. *P<0.05 compared with control group.

Figure 3. Effects of miR-330-5p and ITGA5 on cell proliferation and invasion of GBM cells

(A) Relative cell viabilities detected by MTT assay in mimics and SiITGA5 groups were lower than that in control, NC, and miR-mimics+ITGA5 groups, suggesting that overexpression of miR-330-5p or suppression of ITGA5 could both inhibit cell viability. (B) The colonies in mimics and SiITGA5 groups were obviously less than that in control, NC, and miR-mimics+ITGA5 groups. (C and D) There were fewer cells observed in mimics and SiITGA5 groups, which showed that up-regulation of miR-330-5p and down-regulation of ITGA5 could suppress cell invasiveness. (E and F) Wound healing assay showed the inhibitory effects of up-regulated miR-330-5p and down-regulated ITGA5 on GBM cell migration. *P<0.05 compared with control group.

Figure 4. Effects of miR-330-5p and ITGA5 on cell cycle and apoptosis of GBM cells

(A and B) The cell cycle distribution of U251 cells in different groups showed that cells in mimics or SiITGA5 had higher G₀/G₁ phase and lower S phase compared with cells in control, NC, and miR-mimics+ITGA5 groups. (C and D) The apoptosis rate of U251 cells transfected with miR-330-5p mimics or siRNA-ITGA5 was significantly higher than cells in control, NC, and miR-mimics+ITGA5 groups. *P<0.05 compared with control group.