Silencing long noncoding RNA LINC01138 inhibits aerobic glycolysis to reduce glioma cell proliferation by regulating the microRNA‑375/SP1 axis

Abstract

Glioma is a primary cerebral neoplasm that originates from glial tissue and spreads to the central nervous system. Long noncoding RNAs are known to play a role in glioma cells by regulating cell proliferation, migration and invasion. The aim of the present study was to investigate the mechanism by which long intergenic non‑protein coding RNA (LINC) 01138 affects glycolysis and proliferation in glioma cells via the microRNA (miR)‑375/specificity protein 1 (SP1) axis. LINC01138 expression was assessed in glioma tissues and cells using reverse transcription‑quantitative PCR and the association between LINC01138 and patient clinicopathological features was analyzed. Glucose uptake, lactic acid secretion, cell proliferation, and glycolysis‑related enzyme levels were detected following LINC01138 silencing using CCK‑8, EDU assay and western blot analysis. miR‑375 and SP1 expression levels were also assessed, and the distribution of LINC01138 in the nucleus and cytoplasm was investigated using subcellular fractionation localization. Furthermore, the binding relationships between LINC01138 and miR‑375, and between miR‑375 and SP1 were assessed via dual‑luciferase experiment, RIP and RNA pull‑down assays. Finally, xenograft transplantation models were used to verify the in vitro results. LINC01138 was highly expressed in glioma, which was independent of patient sex or age but was significantly related to tumor diameter, the World Health Organization tumor grade and lymph node metastasis. Silencing LINC01138 significantly reduced glioma glycolysis and cell proliferation. Moreover, LINC01138 acted as a competing endogenous RNA to sponge miR‑375 and promote SP1 expression. miR‑375 inhibition significantly reversed the effect of LINC01138 silencing. In addition, silencing LINC01138 significantly reduced tumor growth in vivo. The present study demonstrated that silencing LINC01138 inhibited aerobic glycolysis and thus reduced glioma cell proliferation, potentially by modulating the miR‑375/SP1 axis.

Keywords: competing endogenous RNA; glycolysis; human glioma; long intergenic non‑protein coding RNA 01138; long noncoding RNA; microRNA‑375; proliferation; specificity protein 1; subcellular localization.

Figure 1.

LINC01138 is highly expressed in human glioma tissues and cells, which is associated with the clinicopathological features of the tumor. (A) LINC01138 expression data in human glioma tissues from The Cancer Genome Atlas database, red represents tumor and grey normal tissue, LINC01138 is significantly highly-expressed in glioma. (B) LINC01138 expression levels in human brain glioma tissues and adjacent normal tissues (n=108) was detected by RT-qPCR. *P<0.05 vs. normal adjacent tissue (paired Student's t-test). (C) LINC01138 expression levels in human brain glioma cells was verified by RT-qPCR. Independent cell experiments were conducted 3 times. *P<0.05 vs. NHAs (one-way ANOVA and Tukey's multiple comparisons test). Data are presented as the mean ± SD. LINC, long intergenic non-protein coding RNA; RT-qPCR, reverse transcription-quantitative PCR; NHAs, normal human astrocytes; num(T), number of tumor tissue samples; num(N), number of normal adjacent tissue samples; GBM, glioblastoma.

Figure 2.

Silencing LINC01138 inhibits human glioma cell proliferation. (A) sh-LINC01138 efficiency in U251MG and TJ905 cells was verified by reverse transcription-quantitative PCR. sh-LINC01138-2 was the most efficient sequence and was chosen for further experiments. U251MG and TJ905 cell proliferation after LINC01138 silencing was detected using the (B) Cell Counting Kit-8 and (C) EdU assays. Independent cell experiments were conducted 3 times. Data are presented as the mean ± SD. *P<0.05 vs. sh-NC (one-way ANOVA and Tukey's multiple comparisons test). LINC, long intergenic non-protein coding RNA; sh, short hairpin RNA; NC, negative control; OD, optical density.

Figure 3.

Silencing LINC01138 inhibits aerobic glycolysis in human glioma cells. (A) Glucose uptake and (B) lactic acid secretion in U251MG and TJ905 cells after LINC01138 silencing was detected by scintillation counter and lactic acid detection kit. (C) Ratio of glucose consumption in the glycolysis pathway in U251MG and TJ905 cells following silencing of LINC01138. *P<0.05 vs. sh-NC (one-way ANOVA and Tukey's multiple comparisons test). (D) Glycolysis-associated protein expression levels in U251MG and TJ905 cells following LINC01138 silencing was verified by western blotting. Independent cell experiments were conducted 3 times. Data are presented as the mean ± SD. *P<0.05 vs. sh-NC (one-way ANOVA and Tukey's multiple comparisons test). LINC, long intergenic non-protein coding RNA; sh, short hairpin RNA; NC, negative control; HK, hexokinase; PK, pyruvate kinase; PFK, phosphofructokinase; GLUT, glucose transporter.

Figure 4.

LINC01138 modulates miR-375 by acting as a competing endogenous RNA. (A) LINC01138 subcellular localization was predicted using lncLocator software. (B) LINC01138 subcellular localization in U251MG cells was verified by the fractionation of nuclear and cytoplasmic RNA, with GAPDH as the cytoplasmic marker and U6 as the nuclear marker. *P<0.05 vs. U6 expressed in the cytoplasm. (C) LINC01138 and miR-375 binding site was predicted by the bioinformatics software RNA22. (D) LINC01138 and miR-375 binding site was confirmed using the dual-luciferase reporter assay. *P<0.05 vs. mimic-NC. (E) The LINC01138 and miR-375 binding relationship was detected by RNA immunoprecipitation. ^#P<0.05 vs. IgG. (F) Binding of miR-375 to LINC01138 was confirmed by RNA pull-down assay. ^&P<0.05 vs. Bio-probe NC. (G) miR-375 expression levels in human glioma tissues and adjacent normal tissues (n=108) was verified by RT-qPCR. *P<0.05 vs. normal adjacent tissues. (H) miR-375 expression levels in human glioma cell lines was determined by RT-qPCR. *P<0.05 vs. NHAs. (I) miR-375 expression levels in U251MG and TJ905 cells following LINC01138 silencing was detected by RT-qPCR. *P<0.05 vs. sh-NC. Data were analyzed using (B and D) two-way ANOVA and Tukey's multiple comparisons test; (E, G and I) independent samples t-test; and (F and H) one-way ANOVA and Tukey's multiple comparisons test. Independent cell experiments were conducted three times. Data are presented as the mean ± SD. LINC, long intergenic non-protein coding RNA; miR, microRNA; NC, negative control; RT-qPCR, reverse transcription-quantitative PCR; NHA normal human astrocyte; sh, short hairpin RNA; WT, wild-type; MUT, mutant.

Figure 5.

miR-375 inhibition reverses the effect of LINC01138 knockdown on aerobic glycolysis and cell proliferation in human glioma. (A) Transfection efficiency of the miR-375 inhibitor was verified by reverse transcription-quantitative PCR. (B) Glucose uptake and (C) lactic acid secretion in U251MG cells were assessed by the scintillation counter and lactic acid detection kit. (D) Ratio of glucose consumption in the glycolysis pathway. (E) U251MG cell proliferation was detected by the Cell Counting Kit-8 assay. Independent cell experiments were conducted 3 times. Data are presented as the mean ± standard deviation. *P<0.05 vs. sh-LINC01138. Data were analyzed using (A-D) one-way ANOVA and Tukey's multiple comparisons test; and (E) two-way ANOVA and Tukey's multiple comparisons test. miR, microRNA; LINC, long intergenic non-protein coding RNA; sh, short hairpin RNA; NC, negative control; OD, optical density; HK, hexokinase.

Figure 6.

LINC01138 can promote SP1 expression by competing with SP1 for binding to miR-375. (A) SP1 expression data in human glioma tissues from The Cancer Genome Atlas database, red represents tumor and grey normal tissue and SP1is highly expressed in tumor tissue. (B) miR-375 and SP1 binding site was predicted using TargetScan software. (C) miR-375 and SP1 binding relationship was confirmed by the dual-luciferase reporter assay. *P<0.05 vs. mimic-NC. (D) SP1 mRNA expression levels in human glioma tissues and adjacent normal brain tissues was determined by RT-qPCR, n=108. *P<0.05 vs. adjacent normal tissues. (E) SP1 mRNA expression levels in NHAs and human brain glioma cell lines were verified by RT-qPCR. *P<0.05 vs. NHAs. (F) SP1 mRNA expression levels in differently transfected cells were measured by RT-qPCR. (G) SP1 protein expression levels in cells were determined by western blotting. *P<0.05 vs. sh-NC. ^#P<0.05 vs. sh-LINC01138 + inhibitor-NC group. Three independent repeated cell experiments were conducted. Data are presented as the mean ± SD. Data were analyzed using (C) two-way ANOVA and Tukey's multiple comparisons test; (D) independent samples t-test; and (E-G) one-way ANOVA and Tukey's multiple comparisons test. miR, microRNA; LINC, long intergenic non-protein coding RNA; SP1, specificity protein 1; NC, negative control; RT-qPCR, reverse transcription-quantitative PCR; NHA, normal human astrocytes; sh, short hairpin RNA; WT, wild-type; MUT, mutant; num(T), number of tumor samples; num(N), number of normal adjacent tissue samples; GBM, glioblastoma.

Figure 7.

Silencing LINC01138 inhibits tumor growth in vivo. (A) Tumor growth curve in nude mice following LINC01138 silencing. (B) Representative images of glioma tumors in nude mice following LINC01138 silencing. (C) Weight of glioma tumors in nude mice following LINC01138 silencing. (D) LINC01138 and miR-375 expression levels in glioma tissues were determined by reverse transcription-quantitative PCR. (E) Specificity protein 1 protein expression levels in glioma tissues were determined by immunohistochemical staining. *P<0.05 vs. sh-NC; n=8. Data were analyzed using (A) two-way ANOVA and Tukey's multiple comparisons test; and (C and E) independent samples t-test; and (D) one-way ANOVA and Tukey's multiple comparisons test. LINC, long intergenic non-protein coding RNA; miR, microRNA; NC, negative control; sh, short hairpin RNA.