PLIN1 suppresses glioma progression through regulating lipid metabolism

Abstract

Glioma is a common and destructive brain tumor, which is highly heterogeneous with poor prognosis. Developing diagnostic and prognostic markers to identify and treat glioma early would significantly improve the therapeutic outcomes. Here, we conducted RNA next-generation sequencing with 33 glioma samples and 15 normal brain samples. We found Perilipin 1 (PLIN1) downregulated in glioma and correlated with poorer outcome. Subsequent experiments revealed that up regulation of PLIN1 led to repressed cell growth and invasion in glioma. Moreover, overexpression of PLIN1 increased lipid accumulation in glioma cells, with increasing expression of lipid biosynthesis related genes and decreasing expression of lipolysis related genes. Mechanically, we revealed that the PI3K/AKT axis could regulate PLIN1 levels in glioma, that inhibition of the activity of PI3K/AKT axis could increase PLIN1 levels in glioma. In conclusion, the dysregulation PI3K/AKT axis led to PLIN1 downregulation and the following tumor proliferation, invasion and lipid metabolism reprogramming in glioma.

Fig. 1. PLIN1 is downregulated and correlated with worse outcome of glioma.

A Differentially expressed genes in 33 glioma tissues (T) compared with 15 normal brain tissues (N). Red: up regulated; Green: down regulated. Fold change > 2 and p < 0.05. B Volcano plots showing genes differentially expressed in 33 glioma tissues (T) compared with 15 normal brain tissues (N). Red: upregulated; Blue: downregulated. Fold change > 2 and p < 0.05. C Hierarchical clustering showing the top 20 upregulated or downregulated genes in glioma samples (T) compared with normal samples (N). Red: up regulated; Green: down regulated. D PLIN1 levels in lower grade glioma and glioblastoma (GBMLGG) dataset from TCGA including 5 normal samples, 670 primary tumor samples as well as 27 recurrent tumor samples were shown. E The OS curve of PLIN1 in lower grade glioma downloaded from the OncoLnc Kaplan Plot website was shown.

Fig. 2. PLIN1 suppresses proliferation and invasion of glioma.

A PLIN1 levels in 30 paired glioma samples and normal adjacent samples by qRT-PCR. B PLIN1 levels in glioma cells were detected by qRT-PCR. C PLIN1 overexpression in glioma cells was confirmed by qRT-PCR. D The intracellular localization and the overexpression of PLIN1 in glioma cells were detected by Immunofluorescence staining. E CCK-8 assay after PLIN1 overexpression. F Colony formation assay after PLIN1 upregulation. Left: represent image of colony formation assay; Right: colony formation number was quantified by ImageJ software. G Transwell assay after PLIN1 upregulation. Left: represent image of Transwell assay; Right: invasive cell number was quantified. H Glioma xenograft models were constructed with U87-PLIN1 overexpressing cell or non-treated U87 cell as control. Left: represents image of xenograft tumors. Right: the tumor weights were measured. I The expression of PLIN1 and Ki67 in xenograft tumors were detected by IHC staining. *P < 0.05, **P < 0.01. The experiment was repeated three times independently.

Fig. 3. Inhibition of PLIN1 enhances proliferation and invasion of glioma.

A Inhibition of PLIN1 in glioma cells was confirmed by qRT-PCR. B Inhibition of PLIN1 in glioma cells was confirmed by western blotting. And the western blotting bands were quantified with ImageJ software (Right). C CCK-8 assay after PLIN1 inhibition. D Colony formation assay after PLIN1 inhibition. Left: represent image of colony formation assay; Right: colony formation number was quantified by ImageJ software. E Transwell assay after PLIN1 inhibition. Left: represent image of Transwell assay; Right: invasive cell number was quantified. F Glioma xenograft models were constructed with SHG-44-PLIN1 knockdown cell or non-treated SHG-44 cell as control. Left: represent image of xenograft tumors. Right: the tumor weights were measured. G The expression of PLIN1 and Ki67 in xenograft tumors were detected by IHC staining. *P < 0.05, **P < 0.01. The experiment was repeated three times independently.

Fig. 4. PLIN1 suppresses glioma progression through regulating lipid metabolism.

A KEGG analysis in the above 33 glioma samples and 15 brain normal samples was shown. B GSEA analysis presented the relationship between glycerophospholipid metabolism and glioma tumorigenesis. C GSEA analysis presented the relationship between fat cell differentiation and glioma tumorigenesis. D Oil red O staining conducted to detect intracellular lipid contents after PLIN1 overexpression in glioma cell lines (Left). Intracellular lipid droplet contents were quantified by ImageJ software (Right). E The intracellular TG levels were detected after PLIN1 overexpression. F The intracellular FFA levels were detected after PLIN1 overexpression. G Lipid metabolism-related genes levels were determined by western blotting. The western blotting bands were quantified with ImageJ software (Right). *P < 0.05, **P < 0.01. The experiment was repeated three times independently.

Fig. 5. The PI3K/AKT axis regulates PLIN1 expression in glioma.

A GO analysis in the above 33 glioma samples and 15 brain normal samples was shown. B Western blotting was performed after PI3K inhibition in glioma cell lines. C The western blotting result was quantified with ImageJ software. *P < 0.05, **P < 0.01. The experiment was repeated three times independently.

Fig. 6. The PI3K/AKT axis regulates glioma progression through PLIN1.

A CCK-8 assay was performed after cell were treated with PI3K inhibitor LY294002, LY294002 plus si-PLIN1 or control. B Colony formation assay was performed after cell were treated with LY294002, LY294002 plus si-PLIN1 or control. C Colony formation number was quantified by ImageJ software. D Transwell assay was performed after cell were treated with LY294002, LY294002 plus si-PLIN1 or control. E Invasive cell number was quantified by ImageJ software. F Oil red O staining conducted to detect intracellular lipid contents after cell were treated with LY294002, LY294002 plus si-PLIN1 or control. G Intracellular lipid droplet contents were quantified by ImageJ software. H The intracellular TG levels were detected after cell were treated with LY294002, LY294002 plus si-PLIN1 or control. I The intracellular FFA levels were detected after cell were treated with LY294002, LY294002 plus si-PLIN1 or control. **P < 0.01. The experiment was repeated three times independently.