Linc00475 promotes the progression of glioma by regulating the miR-141-3p/YAP1 axis

Abstract

Glioma is the most prevalent and lethal primary brain tumour. Abundant long non-coding RNAs ( lncRNAs) are aberrant and play crucial roles in the oncogenesis of glioma. The exact functions of linc00475 in glioma remain blurred. Here, we analysed the expression levels of linc00475 by qRT-PCR and discovered that linc00475 was up-regulated in glioma and predicted a poor prognosis in patients with glioma. Besides, inhibiting linc00475 restrained the progression of glioma in vitro and in vivo. Further experiments confirmed that linc00475 regulated the progression of glioma by acting as a sponge for miR-141-3p. Moreover, we detected the binding sites of linc00475 and miR-141-3p, the YAP1- 3'UTR and miR-141-3p by luciferase reporters. The rescue assays confirmed that inhibiting linc00475 restrained the progression of glioma through the miR-141-3p/YAP1 pathway. Collectively, our research demonstrates the key roles of linc00475 in glioma, which could be a promising therapeutic target.

Keywords: YAP1; ceRNA; glioma; linc00475; miR-141-3p; prognosis.

Figure 1

Linc00475 is over‐expressed in glioma tissues and cells. A, Linc00475 is one of the most up‐regulated lncRNAs in glioma according to the TCGA datasets (Cancer RNA‐Seq Nexus). Columns refer to the samples and rows refer to up‐regulated lncRNAs. The datasets includes 156 glioma samples (1) and 5 normal tissues (2), P < .01. B, qRT‐PCR analysis of linc00475 expression in normal human astrocytes (NHA) and glioma cells (U87 and U251). **P < .01 vs NHA. C, Expression of linc00475 in 15 cases of normal brain tissues (NBTs), 20 cases of low‐grade glioma (LGG) and 20 cases of high grade glioma (HGG). **P < .01 vs NBTs. D and E, Over‐expressed linc00475 was associated with shorter OS in LGG (D) and Glioblastoma (E) (GEPIA). F, Overall Kaplan–Meier survival curve for a cohort of 40 glioma patients, according to the linc00475 expression levels. The linc00475 RNA levels were normalized to those of GAPDH

Figure 2

Linc00475 promotes the progression of glioma cells in vitro. A, Expression of linc00475 in glioma cells after transfection with sh‐linc00475 or the vectors. B, Cell viability of glioma cells was assessed by the CCK‐8 assay after transfection with sh‐linc00475 or the vectors. C, Effect of linc00475 on migration and invasion of glioma cells. D, Western blot analysis of YAP1 and MMP2 expression in the glioma cell lines after transfection with sh‐linc00475. For A‐C, variables are shown as mean ± SD. (n = 3, each group), *P < .05 vs Vector group. Scale bars represent 20 μm

Figure 3

Knock‐down of linc00475 inhibits glioma growth in vivo. A and B, representative images of nude mice bearing transplant tumors (A) and sample tumors (B) from different treatment groups are shown. C, Survival curves of orthotopic xenograft from the different treatment groups (P > .05 vs Vector, n = 8, each group). D and E, The volume (D) and weight (E) of the harvested tumors; variables are shown as mean ± SD. (n = 5, each group), **P < .01 vs Vector group

Figure 4

Linc00475 binds with miR‐141‐3p directly. A, Heatmap of up‐regulated miRNAs after inhibiting linc00475 in U87 glioma cells. B, Expression of miR‐141‐3p in 15 cases of NBTs, 20 cases of LGG and 20 cases of HGG. **P < .01. C, qRT‐PCR analysis of miR‐141‐3p expression in NHA and glioma cells. **P < .01 vs NHA group. D, Linear regression analysis between linc00475 and miR‐141‐3p expression levels in a cohort of 40 glioma tissues. E, Expression of miR‐141‐3p in glioma cells after transfection with sh‐linc00475. *P < .05 vs Vector. F, Expression of linc00475 in glioma cells after changing the expression of miR‐141‐3p. *P < .05. G, The luciferase reporter vectors and the predicted binding sites between linc00475 and miR‐141‐3p. H, Relative luciferase activity in HEK293T cells for the indicated treatments. *P < .05 vs pre‐NC. Variables are shown as mean ± SD (n = 3, each group)

Figure 5

MiR‐141‐3p binds with the YAP1‐3′UTR directly. A, The luciferase reporter vectors and the predicted binding sites between YAP1‐3′UTR and miR‐141‐3p. B, Relative luciferase activity for the indicated treatments. Variables are shown as mean ± SD. (n = 3, each group) *P < .05 vs YAP‐1‐3′UTR‐mut + miR‐141‐3p. C, The Western blot analysis uncovers that inhibiting miR‐141‐3p restores the shRNA2‐induced restrain of YAP1 and MMP2

Figure 6

Linc00475 facilitates the progression of glioma by regulating miR‐141‐3p/YAP1. A, The expression of miR‐141‐3p measured by qPCR in over‐expressed or silenced miR‐141‐3p glioma cells.*P < .05. B, Viability of glioma cells via CCK‐8 assays with sh‐linc00475 was rescued by co‐transfection with shRNA2 and anti‐miR‐141‐3p or co‐transfection with shRNA2 and YAP1. C, Migration and invasion of glioma cells via transwell assays with sh‐linc00475 was rescued by co‐transfection with shRNA2 and anti‐miR‐141‐3p or co‐transfection with shRNA2 and YAP1. Variables are shown as mean ± SD. (n = 3, each group), *P < .05 vs shRNA2. Scale bars represented 20 μm