LINC01198 promotes proliferation and temozolomide resistance in a NEDD4-1-dependent manner, repressing PTEN expression in glioma

Figures

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  Figure 1. LINC01198 is overexpressed in human glioma. (A) Expression level of LINC01198 was determined and normalized to GAPDH in 90 pairs of glioma tissues and their corresponding adjacent normal tissues by qRT-PCR. (B) The patients were divided into the tumor size ≤3 cm and tumor size >3 cm groups. The diagram shows the LINC01198 expression in each group. P = 0.001. (C) The patients were divided into the stage I-II and III-IV groups. The diagram shows the LINC01198 expression in each group. P = 0.006. (D and E) Glioma patients were divided into the LINC01198High group and LINC01198Low group according to the result of qRT-PCR. Survival and recurrence curves were constructed using the log-rank test. The results show the OS and recurrence for glioma patients with high LINC01198 expression and those with low LINC01198 expression.

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  Figure 2. LINC01198 promotes glioma proliferation and resistance to temozolomide. (A) LINC01198 expression was examined in several glioma cell lines, normal brain tissues, and glioma tissues using qRT-PCR. GAPDH was used as a control for loading. (B and C) LINC01198 expression in glioma cells was modified by shRNA interference and cDNA transfection. (D) Growth curve showing the proliferation activities of glioma cell transfectants in vitro. (E) IC50 value of temozolomide on glioma cells. Cells were treated with different concentrations of temozolomide for 72 hours. (F) Antitumor effect of temozolomide on glioma xenografts in an established model. (G) The data were expressed as percentage inhibition of tumor growth. (H) The survival time of mice bearing glioma subcutaneous xenograft received temozolomide therapy. Data are presented as the mean ± SD, n=3. *P < 0.05, **P < 0.01.

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  Figure 3. LINC01198 functions as a scaffold for NEDD4-1/PTEN to regulate PTEN expression in glioma cells. (A) Predicted interaction probabilities of LINC01198 and RNA-binding proteins via a RNA–protein interaction prediction tool. (B) RIP assays of LINC01198 binding to indicated proteins in glioma U87 cell extracts. (C) RNA pulldown was used to examine the association of LINC01198 and NEDD4-1/PTEN. (D) The LINC01198 gene was cut into LINC01198-D1(1-500 nt) and -D2(501-955 nt), and their overexpression vectors were constructed and transfected into U87 cells. (E) RIP assays of LINC01198-D1/D2 binding to indicated proteins in glioma U87 cell extracts. (F) Biotinylated LINC01198-D1/D2 RNAs were incubated with U873 cell lysates, and western blotting analysis was performed to evaluate the specific association between them and NEDD4-1/PTEN.

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  Figure 4. LINC01198 increased NEDD4-1-induced PTEN inhibition in glioma cells. (A) Forced LINC01198 expression inhibited PTEN expression in glioma cells. (B) Protein expression levels of PTEN were increased in glioma cells treated with LINC01198 shRNA. (C) Forced LINC01198 expression did not affect NEDD4-1 expression in glioma cells. (D) Protein expression levels of NEDD4-1 did not change in glioma cells treated with LINC01198 shRNA. (E) Forced LINC01198 expression did not inhibit PTEN expression in CRISPR/cas9-induced NEDD4-1-knockout glioma cells. (F) Protein expression levels of PTEN were not increased in CRISPR/cas9-induced NEDD4-1-knockout glioma cells treated with LINC01198 shRNA. (G and H). A negative correlation between LINC01198 and PTEN was observed in tumor tissues at the protein level (r = 0.501; P = 0.013), but this correlation was not observed at the mRNA level (r = 0.296; P > 0.05).

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  Figure 5. NEDD4-1 reversed LINC01198-induced glioma proliferation and resistance to temozolomide. (A) Forced LINC01198 expression did not increase cell proliferation in NEDD4-1-knockout glioma cells. (B) Forced LINC01198 expression did not promote resistance to temozolomide in NEDD4-1-knockout glioma cells. (C) Reduced LINC01198 expression did not inhibit cell proliferation in NEDD4-1-knockout glioma cells. (D) Reduced LINC01198 expression did not inhibit resistance to temozolomide in NEDD4-1-knockout glioma cells. (E) PTEN expression in LINC01198-overexpressing glioma cells was modified by cDNA transfection. (F) recovered PTEN expression reversed LINC01198-induced tumor growth in glioma. Data are presented as the mean±S.D, n=3.

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  Figure 6. LINC01198 enhances AKT activity through regulation of the NEDD4-1/PTEN axis in glioma cells. (A) Forced LINC01198 expression upregulated p-AKT expression in glioma cells. (B) Protein expression levels of p-AKT were reduced in glioma cells treated with LINC01198 shRNA. (C) LINC01198 knockdown inhibited the interplay between NEDD4-1 and PTEN in glioma cells. (D) Forced LINC01198 expression did not upregulate p-AKT expression in CRISPR/cas9-induced NEDD4-1-knockout glioma cells.

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  Figure 7. LINC01198 induces temozolomide resistance in glioma patients. (A) Recurrent glioma patients were divided into the LINC01198High group and LINC01198Low group according to the result of qRT-PCR. (B) Comparison of overall survival curves for temozolomide-treated patients with high LINC01198 expression and those with low LINC01198 expression.

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  Figure 8. Working model: LINC01198 overexpression promotes the proliferation and temozolomide resistance of glioma cells.