Analysis of differential expression of GABARAP family members in Glioblastoma

Description

Our study is based on the analysis of the differential expression of GABARAP family members in glioblastoma (GBM). We investigated the expression of GABARAP’s (GABARAP, GABARAPL1, and GABARAPL2) and MAP1LC3’s (MAP1LC3A, MAP1LC3B, and MAP1LC3C) in GBM and Low-grade glioma (LGG) patient samples. We found that the GABARAP gene was highly expressed at transcript levels in the GBM patient sample as compared to other members of GABARAP and MAP1LC3’s. Since GABARAP’s and MAP1LC3’s genes are involved in the autophagy initiation and the autophagosome formation. So, we investigated the expression of these family members after autophagy regulation in GBM cell lines: U87MG and LN229. We used chloroquine (CQ) and Early Balanced Salt Solution (EBSS) for inhibition and initiation of autophagy. Chloroquine is a late-stage autophagy inhibitor while EBSS is a starvation induce media. After autophagy inhibition, the non-lipidated form of GABARAP (I) was increased. However, no changes were observed in GABARAPL1 and GABARAPL2 in both cell lines. Non-lipidated form of GABARAPL2 (II) showed increased expression in EBSS conditions. Temozolomide (TMZ) is a DNA alkylating agent used in the clinical treatment of newly diagnosed GBM. Interestingly, only the lipidated form of GABARAP genes was significantly highly expressed after TMZ concentration-dependent (200µM and 400 µM) and time-dependent treatments (24hrs and 48hr). We have got the same findings after treatment with IC50 concentration of TMZ in U87MG. We also analyzed that MAP1LC3A expression decreases after TMZ treatment. These outcomes conclude that ATG8 genes behave differently in response to TMZ treatment. We further investigated the role of GABARAP’s in GBM by its knockdown and overexpression. We performed an MTT assay to check the TMZ sensitivity after GABARAP’s knockdown and overexpression. We observed that TMZ sensitivity is decreased with GABARAP knockdown but not with knockdown of GABARAPL1 and GABARAPL2. After GABARAP knockdown the IC50 concentration of TMZ was significantly increased but the opposite result was seen after GABARAP overexpression. We also observed that GABARAP overexpression induced p53 expression upon TMZ treatment. P53 is a tumor suppressor gene and it shows a positive correlation with GABARAP. We’ve also checked the expression of P53 after GABARAP’s knockdown which was interestingly, decreased. Furthermore, we also found that a combined treatment of TMZ and CQ with IC50 concentrations increases the P53 expression. Our study demonstrated that the overexpression of GABARAP increased the sensitivity of TMZ with synergistic increased in P53. The differential role of GABARAPs can be useful for better therapeutic approaches in GBM.

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