LINC01116 promotes the proliferation and invasion of glioma by regulating the microRNA‑744‑5p‑MDM2‑p53 axis

Abstract

Long non‑coding RNAs (lncRNAs) have been implicated in the development and progression of tumors. However, the roles and underlying mechanisms of long intergenic non‑protein coding RNA 1116 (LINC01116), a member of the lncRNA family, in glioma progression are largely unclear. The expression of LINC01116 and microRNA (miR)‑744‑5p in glioma tissues and cells was detected by reverse transcription‑quantitative PCR. The influences of LINC01116 or miR‑744‑5p on cell proliferation and invasion were evaluated by Cell Counting Kit‑8, colony formation and Transwell assays, and western blotting was used to detect the expression of p53 pathway proteins. A dual‑luciferase reporter system was used to locate common binding sites between miR‑744‑5p and LINC01116 or the 3' untranslated region of E3 ubiquitin‑protein ligase Mdm2 (MDM2). RNA immunoprecipitation was used to determine the interactions between RNAs and proteins. Moreover, a xenograft mouse model was constructed to investigate the effects of LINC01116 in vivo, followed by a TdT‑mediated dUTP nick end labeling assay to determine the degree of apoptosis in nude mouse tumors. LINC01116 was found to be highly expressed in glioma tissues, which was associated with a malignant phenotype. LINC01116 promoted the proliferation and invasiveness of glioma cells, and inhibited the p53 pathway by preserving the expression of MDM2 mRNA via miR‑744‑5p sponging. Furthermore, a low degree of miR‑744‑5p expression was observed in glioma tissues, which was negatively associated with the expression of LINC01116. Overexpression of miR‑744‑5p inhibited the proliferation and invasiveness of glioma cells, which was rescued by LINC01116. Finally, LINC01116 knockdown inhibited tumor growth in nude mice. In conclusion, LINC01116 is aberrantly expressed and promotes the progression of glioma by regulating the miR‑744‑5p‑MDM2‑p53 pathway. In future, targeting LINC01116 may therefore be a potential therapeutic approach for patients with glioma.

Keywords: glioma; long intergenic non‑protein coding RNA 1116; microRNA‑744‑5p; MDM2; p53 pathway; competing endogenous RNA.

Figure 1.

LINC01116 expression is upregulated in glioma and predicts poor prognosis. (A) Relative expression of LINC01116 in GBM and LGG was analyzed using the GEPIA platform. Compared with normal brain tissues, GBM tissues exhibited higher expression levels of LINC01116. (B-D) Overall survival analysis based on LINC01116 expression levels in different grade glioma tissues was also analyzed on the GEPIA platform. Regardless of glioma grade (i.e. total glioma patients), the survival times of patients with high LINC01116 were significantly shorter than those with low LINC01116 expression. (E) Relative expression of LINC01116 in 46 fresh glioma tissues. Both II–III grade gliomas (n=17) and GBMs (n=29) exhibited significantly higher LINC01116 expression levels than normal brain tissues (n=4). (F) Expression of LINC01116 was positively correlated with the percentage of Ki-67-positive cells in glioma tissues (n=46). *P<0.05, ***P<0.001. LINC01116, long intergenic non-protein coding RNA 1116; GBM, glioblastoma; LGG, lower grade glioma; GEPIA, Gene Expression Profiling Interactive Analysis.

Figure 2.

LINC01116 promotes the proliferation and invasiveness of glioma. (A) Relative expression of LINC01116 in HEB and glioma cell lines (U87, A172 and Shg44). Stable (B) LINC01116 KD and (C) LINC01116 OE cell lines were constructed using Shg44 and A172 cells, respectively. Relative LINC01116 expression in stable KD or OE cells was assessed by reverse transcription-quantitative PCR. Cell Counting Kit-8 assays showed that (D) LINC01116 KD decreased Shg44 cell proliferation, whereas (E) LINC01116 OE promoted the proliferation of A172 cells. (F) Effects of LINC01116 KD and OE on the colony formation and invasive abilities of Shg44 and A172 cells respectively. Histograms indicate the relative numbers of colonies and cells. *P<0.05, **P<0.01 and ***P<0.001 vs. NC. LINC01116, long intergenic non-protein coding RNA 1116; KD, knockdown; OE, overexpression; NC, negative control.

Figure 3.

LINC01116 mediates the MDM2-p53 pathway in glioma. (A) Gene Expression Profiling Interactive Analysis revealed that LINC01116 levels negatively correlate with the mRNA levels of p53 target genes BAK1, BAX, CDKN1A and GADD45A in GBM tissues (n=81). (B and C) LINC01116 regulated the mRNA expression of MDM2, BAK1 and GADD45A, but not TP53 in glioma cell lines. (D) Protein expression of MDM2, p53, BAK1 and GADD45A were regulated by LINC01116 in glioma cells. (E) Histogram indicating the relative protein levels in (D). (F) LINC01116 regulated the p53 pathway in A172 cells, which was dependent on the presence of MDM2. (G) Histogram indicating the relative protein levels in (F). *P<0.05, **P<0.01 and ***P<0.001 vs NC. LINC01116, long intergenic non-protein coding RNA 01116; MDM2, E3 ubiquitin-protein ligase Mdm2; KD, knockdown; OE, overexpression; NC, negative control; CDKN1A, cyclin-dependent kinase inhibitor 1; GADD45A, growth arrest and DNA damage-inducible protein GADD45 α.

Figure 4.

LINC01116 increases MDM2 mRNA levels by sponging miR-744-5p in glioma. (A) LINC01116 negatively regulated the expression of miR-744-5p in Shg44 and A172 cells. (B) mRNA expression of LINC01116 and MDM2 were downregulated by miR-744-5p mimics in Shg44 cells, and upregulated by the miR-744-5p inhibitor in A172 cells. (C) LINC01116 OE partially rescued the expression of MDM2 mRNA and protein downregulated by miR-744-5p mimics in A172 cells. (D) miR-744-5p inhibitors partially rescued MDM2 mRNA and protein expression downregulated by LINC01116 KD in Shg44 cells. (E) Predicted binding sites between miR-744-5p and LINC01116 or the MDM2 mRNA 3′-UTR in WT and MUT sequences. (F) miR-744-5p significantly reduced the luciferase activity of WT LINC01116 in 293T cells, but not the MUT sequence, and the reduction in luciferase activity was rescued by LINC01116 OE. (G) Luciferase activity of the WT MDM2 3′-UTR, but not the MUT, was significantly reduced by miR-744-5p mimics in 293T cells. However, miR-744-5p inhibitor did not significantly change the luciferase activity of LINC01116 WT or MUTplasmids. (H) RNA immunoprecipitation assays showed that both LINC01116 and miR-744-5p could bind Ago2. *P<0.05, **P<0.01 and ***P<0.001. LINC01116, long intergenic non-protein coding RNA 01116; MDM2, E3 ubiquitin-protein ligase Mdm2; miR, microRNA; KD, knockdown; OE, overexpression; NC, negative control; UTR, untranslated region; WT, wild-type; MUT, mutant.

Figure 5.

miR-744-5p inhibits the proliferation and invasiveness of glioma and reverses the biological functions of LINC01116. (A) miR-744-5p was significantly downregulated at different glioma grades. (B) miR-744-5p expression was negatively correlated with the percentage of Ki-67 in glioma tissues. (C) Relative levels of miR-744-5p in different glioma cell lines. Cellular proliferation was (D) reduced by miR-744-5p mimics in Shg44 cells, and (E) upregulated by the miR-744-5p inhibitor in A172 cells. (F) miR-744-5p expression was negatively correlated with LINC01116 expression in glioma tissues. (G) Colony formation and invasive ability of Shg44 cells were inhibited by miR-744-5p mimics, which was reversed by OE of LINC01116. (H) Histograms indicate the relative number of colonies and cells. (I) miR-744-5p inhibitor promoted the colony formation and invasive ability of A172 cells and partially restored these behaviors inhibited by LINC01116 KD. (J) Histograms indicate the relative number of colonies and cells. *P<0.05, **P<0.01 and ***P<0.001 vs NC. miR, microRNA; LINC01116, long intergenic non-protein coding RNA 1116; KD, knockdown; OE, overexpression; NC, negative control.

Figure 6.

LINC01116 promotes gliomagenesis in vivo. (A) LINC01116 KD inhibited the growth of Shg44 cell tumors. Histogram indicating the mean weight of the xenograft tumors (n=5). Line chart of tumor growth curves (n=5). (B) mRNA expression levels of LINC01116, miR-744-5p, MDM2 and TP53 in the indicated xenograft tumors. (C) Protein expression of BAK1, GADD45A, MDM2 and p53 in xenograft tumors. (D) TUNEL assays were performed to detect glioma cell apoptosis in the xenograft tumors. Histogram indicating the percentage of apoptotic cells in the xenograft tumors (n=5). Magnification, ×20. **P<0.01 and ***P<0.001 vs NC. LINC01116, long intergenic non-protein coding RNA 01116; miR, microRNA; KD, knockdown; NC, negative control; GADD45A, growth arrest and DNA damage-inducible protein GADD45 α; MDM2, E3 ubiquitin-protein ligase Mdm2.