ZEB2 mediates multiple pathways regulating cell proliferation, migration, invasion, and apoptosis in glioma

Abstract

Background: The aim of the present study was to analyze the expression of Zinc finger E-box Binding homeobox 2 (ZEB2) in glioma and to explore the molecular mechanisms of ZEB2 that regulate cell proliferation, migration, invasion, and apoptosis.

Methodology/principal findings: Expression of ZEB2 in 90 clinicopathologically characterized glioma patients was analyzed by immunohistochemistry. Furthermore, siRNA targeting ZEB2 was transfected into U251 and U87 glioma cell lines in vitro and proliferation, migration, invasion, and apoptosis were examined separately by MTT assay, Transwell chamber assay, flow cytometry, and western blot.

Results: The expression level of ZEB2 protein was significantly increased in glioma tissues compared to normal brain tissues (P<0.001). In addition, high levels of ZEB2 protein were positively correlated with pathology grade classification (P = 0.024) of glioma patients. Knockdown of ZEB2 by siRNA suppressed cell proliferation, migration and invasion, as well as induced cell apoptosis in glioma cells. Furthermore, ZEB2 downregulation was accompanied by decreased expression of CDK4/6, Cyclin D1, Cyclin E, E2F1, and c-myc, while p15 and p21 were upregulated. Lowered expression of ZEB2 enhanced E-cadherin levels but also inhibited β-Catenin, Vimentin, N-cadherin, and Snail expression. Several apoptosis-related regulators such as Caspase-3, Caspase-6, Caspase-9, and Cleaved-PARP were activated while PARP was inhibited after ZEB2 siRNA treatment.

Conclusion: Overexpression of ZEB2 is an unfavorable factor that may facilitate glioma progression. Knockdown ZEB2 expression by siRNA suppressed cell proliferation, migration, invasion and promoted cell apoptosis in glioma cells.

Figure 1. Expression of ZEB2 in glioma and normal brain tissues. A and B.

Weak expression of ZEB2 in normal brain tissues. C. Weak expression of ZEB2 in glioma samples. D. Medium expression of ZEB2 in glioma samples. E and F. Strong staining of ZEB2 in glioma samples. (Original magnification 400×).

Figure 2. Effect of siRNA interference on ZEB2 expression in human glioma cell lines U251 and U87.

Different treatments included negative control (NC) and siZEB2-transfected groups (siZEB2). A. RT-PCR shows transcriptional levels of the ZEB2 gene 48 hr post transfection and ARF was used as a loading control. The arbitrary units were plotted using mean ± SD of at least three individual repetitions. B. Western blot showing protein expression levels in NC and siZEB2 treatments. At 72 hr post transfection, cells were harvested and whole cell lysates prepared using RIPA buffer. The representative image of three different repetitions is shown. β-actin served as a loading control. Bar graph shows the relative expression of protein among the groups. Data are presented as mean ± SD for three independent experiments. C. Immunofluorescence study using blinded analysis showing the expression of ZEB2 in NC and siZEB2-treated U251 and U87 cells at 48 hr post-transfection (Original magnification 1000×). *P<0.05, statistically significant difference.

Figure 3. Downregulation of ZEB2 inhibits glioma cell proliferation, migration and invasion in vitro. A.

Effect of ZEB2 knockdown on U251 and U87 cell proliferation as measured by MTT assay. Absorbance was read at 490 nm with averages from triplicate wells. B and C. Downregulation of ZEB2 dramatically reduced U251 and U87 cell migration and invasion in vitro. Data are presented as mean ± SD for three independent experiments (Original magnification 400×). *P<0.05, statistically significant difference.

Figure 4. ZEB2 downregulation promotes expression of E-cadherin but suppresses EMT progression in glioma cells.

ZEB2 reduction enhanced expression of E-cadherin and expression level changes of N-cadherin, Snail, β-Catenin and Vimentin in U251 and U87 cells at 72 hr post-transfection. β-actin was used as a loading control. Bar graph shows the relative expression of protein among the groups. Data are presented were presented as mean ± SD for three independent experiments. *P<0.05, statistically significant difference.

Figure 5. Immunofluorescence of EMT related regulators in NC and siZEB2-treated U251 and U87 cells at 48 hr post-transfection.

High expression of β-Catenin and Vimentin and low E-cadherin expression in U251 and U87 cells was observed. After transfection with siZEB2 in both glioma cell lines, the expression of β-Catenin and Vimentin were decreased while E-cadherin was increased. (Original magnification 400×).

Figure 6. Downregulation of ZEB2 expression induced cell cycle arrest at G1/S phase by regulating cell cycle-related genes.

A. The cell cycle distribution in siZEB2 treated and NC groups of U251 and U87 glioma cells by FACS Caliber cytometry. Histograms showing G1/S phase arrest and a decline in S phase in U251 and U87 cells after knocking down ZEB2 expression through targeted siRNA transfection. B and C. The protein levels of several key cell cycle regulators was analyzed by western blot. Reduced ZEB2 expression significantly inhibited cell cycle progression in U251 and U87 glioma cells. β-actin was used as a loading control. Bar graph shows the relative expression of protein among the groups. Data are presented were presented as mean ± SD for three independent experiments. *P<0.05, statistically significant difference.

Figure 7. ZEB2 downregulation induces apoptosis by the activation of Caspase-3 in glioma cells.

A. Apoptosis in U251 and U87 cells was measured by Annexin V-FITC/propidium iodide (PI) staining following siZEB2 or control treatment. Early apoptotic cell populations were significantly increased (P<0.01) after siZEB2 transfection. B. Western blot analysis for antiapoptotic PARP and effector Caspase-3, Caspase-6 and Caspase-9. Decreased PARP and increased caspase-3,-6,-9, and Cleaved PARP expression were observed in U251 and U87 cells after ZEB2 downregulation at 72 hr post transfection. β-actin was used as a loading control. Bar graph shows the relative expression of protein among the groups. Data are presented were presented as mean ± SD for three independent experiments. *P<0.05, statistically significant difference.