RNA interference targeting EphA2 inhibits proliferation, induces apoptosis, and cooperates with cytotoxic drugs in human glioma cells.
BACKGROUND
Overexpression of EphA2 was detected in low- and high-grade glioma. To examine the role of EphA2 in human glioma cells, we studied its effects on proliferation and apoptosis using gene silencing through RNA interference.


METHODS
One siRNA targeting EphA2 gene was synthesized in vitro and was transfected into the glioma U251n cells. Expression of EphA2 proteins was detected by Western blots and immunofluorescence. Cell apoptosis and mitochondrial membrane potential were analyzed by flow cytometry and annexin-V/fluorescein isothiocyanate/propidium iodide, respectively. Caspase-3 activity was measured by a spectrofluorometer. MTT assay was used to examine changes in cell proliferation.


RESULTS
After treatment with sequence-specific siRNA targeting EphA2, the protein level of the transfected group decreased significantly. As compared to non-siRNA transfected cells, the transfected group showed lower proliferation, higher apoptosis, and loss of mitochondrial membrane potential. Caspase-3 activity increased in cells treated with siRNA and downregulated when treated with caspase-3 inhibitor. And the effects were clearly additive when siRNA transfected cells treated with the anticancer agents.


CONCLUSIONS
The results suggest that EphA2-siRNA inhibit U251n cell proliferation and induce their apoptosis. It is possible that EphA2 via mitochondrial and caspase-3 inhibits U251n cell apoptosis. And EphA2-siRNA transfection enhances U251n cells' sensitivity to chemotherapy. EphA2 may be an effective therapeutic target in patients with glioma. Silencing the receptor EphA2 gene is a novel approach for the containment of growth and migration of tumor in patients with malignant glioma.
Zhou
Zhangming
Z
Department of Neurosurgery, Zhongnan Hospital of Wuhan University, Wuhan 430071, China. zhangmingzhou2010@126.com
Yuan
Xianhou
X
Li
Zhiqiang
Z
Tu
Hanjun
H
Li
Dongsheng
D
Qing
Jun
J
Wang
Hui
H
Zhang
Li
L
D000970:Antineoplastic Agents / Q000627:therapeutic use*; D017209:Apoptosis; D018929:Cell Culture Techniques; D049109:Cell Proliferation; D005909:Glioblastoma / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology*; D006801:Humans; D053078:Membrane Potential, Mitochondrial; D034622:RNA Interference; D034741:RNA, Small Interfering; D036104:Receptor, EphA2 / Q000502:physiology*; D014162:Transfection; D014407:Tumor Cells, Cultured
D000970:Antineoplastic Agents; D034741:RNA, Small Interfering; D036104:Receptor, EphA2