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. 2021 Feb 9;26(4):901.
doi: 10.3390/molecules26040901.

Silencing ZEB2 Induces Apoptosis and Reduces Viability in Glioblastoma Cell Lines

Sahar Safaee  1 , Masoumeh Fardi  1   2 , Nima Hemmat  1 , Neda Khosravi  1 , Afshin Derakhshani  1   3 , Nicola Silvestris  3   4 , Behzad Baradaran  1
Affiliations

Silencing ZEB2 Induces Apoptosis and Reduces Viability in Glioblastoma Cell Lines

Sahar Safaee et al. Molecules. .

Abstract

Background: Glioma is an aggressive type of brain tumor that originated from neuroglia cells, accounts for about 80% of all malignant brain tumors. Glioma aggressiveness has been associated with extreme cell proliferation, invasion of malignant cells, and resistance to chemotherapies. Due to resistance to common therapies, glioma affected patients' survival has not been remarkably improved. ZEB2 (SIP1) is a critical transcriptional regulator with various functions during embryonic development and wound healing that has abnormal expression in different malignancies, including brain tumors. ZEB2 overexpression in brain tumors is attributed to an unfavorable state of the malignancy. Therefore, we aimed to investigate some functions of ZEB2 in two different glioblastoma U87 and U373 cell lines.

Methods: In this study, we investigated the effect of ZEB2 knocking down on the apoptosis, cell cycle, cytotoxicity, scratch test of the two malignant brain tumor cell lines U87 and U373. Besides, we investigated possible proteins and microRNA, SMAD2, SMAD5, and miR-214, which interact with ZEB2 via in situ analysis. Then we evaluated candidate gene expression after ZEB2-specific knocking down.

Results: We found that ZEB2 suppression induced apoptosis in U87 and U373 cell lines. Besides, it had cytotoxic effects on both cell lines and reduced cell migration. Cell cycle analysis showed cell cycle arrest in G0/G1 and apoptosis induction in U87 and U373 cell lines receptively. Also, we have found that SAMAD2/5 expression was reduced after ZEB2-siRNA transfection and miR-214 upregulated after transfection.

Conclusions: In line with previous investigations, our results indicated a critical oncogenic role for ZEB2 overexpression in brain glioma tumors. These properties make ZEB2 an essential molecule for further studies in the treatment of glioma cancer.

Keywords: TGF-β pathway; ZEB2; apoptosis; glioblastoma; siRNA.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
ZEB2-siRNA significantly downregulated ZEB2 in both U87 and U373 cell lines. The results are expressed in comparison to the negative control (transfected with non-target siRNA) and non-transfected groups (control group). The results are shown as mean (M) ± SD (** p < 0.01, *** p < 0.001).
Figure 2
Figure 2
Schematic view of the GSE4290 dataset result after analyzing with GEO2R for the most common genes involved in glioblastoma. It was found that 23 genes are contributed to glioblastoma progression.
Figure 3
Figure 3
MiRwalk results show that miR-214-3p/ZEB2/SMAD-dependent TGF-β axis has an important role in glioblastoma induction or progression.
Figure 4
Figure 4
The results show that ZEB2 suppression is accompanied by significant downregulation of SMAD2 and SMAD5 in both U87 and U373 cells. In comparison, the expression of miR-214-3p in both cell lines was increased after ZEB2 suppression. Our results are presented in comparison to non-transfected group results (control group). Data are shown mean (M) ± SD (* p < 0.05, *** p < 0.001, **** p < 0.0001).
Figure 5
Figure 5
Bar graphs depicting ZEB2 suppression effect on the viability of glioma cells using an MTT kit developed by Sigma Aldrich. The results suggested that compared to control the transfection of U87 and U373 cells by different concentrations of ZEB2-siRNA (60–80 pmol/µL) after 48 from transfection significantly reduced cell proliferation and viability at 60 pmol/µL. In addition, scrambled siRNA had no cytotoxic effect on the cell’s viability (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001)
Figure 6
Figure 6
Effect of ZEB2 knockdown on the cell cycle distribution of U87 and U373 cell lines. (A) ZEB2-transfection increased arrest in the G0/G1 phase leading to fewer the other checkpoints in comparison to control in U373 cells. (B) Transfection induced DNA damage and apoptosis in U87 cell line. * p < 0.05, ** p < 0.01.
Figure 7
Figure 7
Flow cytometry test results by Annexin V-FITC/PI staining indicated that apoptosis was significantly induced in U87 and U373 cells compared to the corresponding control groups following ZEB2-siRNA transfection (** p < 0.01). All results are presented as Mean ± SD.
Figure 8
Figure 8
Our results showed that ZEB2 suppression attenuated the migration of glioblastoma cells. (A,B) Wound scratch assay showing that non-transfected cells (control group) have a significantly greater migratory ability than transfected glioblastoma cells with ZEB2-specific siRNA. The bar graph represents the quantity of migrated cells per field (*** p < 0.001, **** p < 0.0001).
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