Long Non-coding RNA TUSC7, a Target of miR-23b, Plays Tumor-Suppressing Roles in Human Gliomas

Abstract

Tumour suppressor candidate 7 (TUSC7) is a novel tumor suppressor gene generating long non-coding RNA (lncRNAs) in several types of human cancers. The expression and function of TUSC7 in human brain glioma has yet to be elucidated. In this study, TUSC7 was poorly expressed in tissues and cell lines of glioma, and the lower expression was correlated with glioma of the worse histological grade. Moreover, TUSC7 is a prognostic biomarker of glioma patients. Up-regulation of TUSC7 suppressed cellular proliferation and invasion of glioma cells, and accelerated cellular apoptosis. Bioinformatics analysis showed that TUSC7 specifically binds to miR-23b. MiR-23b was up-regulated in glioma and negatively correlated with the expression of TUSC7. The miR-23b expression was inhibited remarkably by the upregulation of TUSC7 and the reciprocal inhibition was determined between TUSC7 and miR-23b.RNA pull-down and luciferase reporter assays were used to validate the sequence-specific correlation between miR-23b and TUSC7. TUSC7 inhibited the proliferation, migration and invasion of glioma cells and promoted cellular apoptosis largely bypassing miR-23b. We conclude that the lncRNA TUSC7 acted as a tumor suppressor gene negatively regulated by miR-23b, suggesting a novel therapeutic strategy against gliomas.

Keywords: TUSC7; glioma; mir-23b; noncoding RNA; tumor suppress gene.

FIGURE 1

LncRNA microarray analysis of total RNA isolated from five glioma tissues and corresponding adjacent normal brain tissues. 1–5: glioma, 1p–5p: normal.

FIGURE 2

(A) Compared with normal brain tissues (NBT) and normal human astrocytes (NHA) cells, TUSC7 was poorly expressed in human glioma tissues and cell lines. (B) Glioma tissues with worse histological grade exhibited decreased TUSC7 expression. (C) Kaplan–Meier analysis indicated that lower TUSC7 expression was associated with a significantly shorter survival time than higher TUSC7 expression. Data are presented as mean ± SD (n = 5, each group). ^∗P < 0.05 vs. NBT or NHA group.

FIGURE 3

(A) TUSC7 expression was up-regulated significant by transfection of pE-TUSC7 in U251 and U87 cell lines. (B,C) TUSC7 over-expression inhibited cell viability and advanced apoptosis of U251 and U87 cells. (D) TUSC7 over-expression impeded the migration and invasion of U251 and U87 cells, Scale bars represent 100 μm.^∗P < 0.05 vs. Blank and NC group.

FIGURE 4

(A) Putative binding sites of miR-23b within TUSC7 mRNA, and the sequences of wild-type and mutant-type vectors. (B) MiR-23b was highly expressed in human glioma tissues and cell lines. (C) Compared with control groups, agomir-23b increased the expression of miR-23b in U251 and U87 cells, antagomir-23b inhibited miR-23b expression. (D) The relative luciferase activities were inhibited in the HEK-293 cells co-transfected with wild-type vector and p agomir-23b, and not with the mutant-type vector. Firefly luciferase activity was normalized to Renilla luciferase. (E) Detection of TUSC7 mRNA using qRT-PCR in the sample pulled down by biotinylated miR-23b. (F) Detection of miR-23b mRNA using qRT-PCR in the sample pulled down by biotinylated TUSC7. (G) MiR-23b expression was down-regulated significantly by TUSC7 over-expression in U251 and U87 cell lines. (H,I) Association of TUSC7 and miR-23b with Ago2 in U251 and U87 cells. Cellular lysates from U251 and U87 cells were used for RIP with antibody against Ago2. TUSC7 and miR-23b expression levels were detected using qRT-PCR. ^∗P < 0.05.

FIGURE 5

(A,B) Mir-23b down-regulation inhibited cell viability and advanced apoptosis of U251 and U87 cells. (C) Mir-23b down-regulation disrupted the migration and invasion of U251 and U87 cells, Scale bars represent 100 μm.^∗P < 0.05 vs. Blank and NC group.

FIGURE 6

Over-expression of miR-23b partly reversed TUSC7-induced inhibition of proliferation (A), migration and invasion (B), and partly reversed TUSC7-induced apoptosis (C) in U251 and U87 cells. Scale bars represent 100 μm. ^∗P < 0.05, ^∗∗P < 0.01.