PTP-MEG2 Inhibits The Malignant Progression Of Human Astrocytoma Through Dephosphorylating EGFR And STAT3

Objective: Astrocytoma is the most common type of brain tumor.The median survival of patients with high grade astrocytoma is still less than one year,although various comprehensive treatments including surgery,radiotherapy and chemotherapy are available.There is a research showing that the mutation,over-amplification and activation of epidermal growth factor receptor(EGFR)is closely related to the occurrence and development of astrocytoma.STAT3,as a downstream target protein of EGFR,is over-activated in astrocytomas.This pathological change is closely related to the proliferation and invasion of astrocytoma.In the EGFR / STAT3 signaling pathway,there are lots of relative studies on the members of the protein tyrosine kinases(PTKs)family.However,few studies have reported on the protein tyrosine phosphatases(PTPs)which mediate the dephosphorylation of p-EGFR.In recent years,the research on PTP-MEG2(PTPN9)has gradually received attention.PTP-MEG2 was first found in human megakaryocyte leukemia cells MEG-01 and human umbilical vein endothelial cells in 1992.It is widely expressed in the human body.Studies have shown that PTP-MEG2 is involved in the malignant progression of breast cancer and liver cancer.At present,neither domestic nor foreign research has dealt with the role of PTP-MEG2 in EGF/EGFR signaling in astrocytomas.Methods:134 cases of astrocytoma-embedded tissue with complete prognostic information and 6 cases of normal brain tissue due to intracranial hypertension were collected.The protein expression level of PTP-MEG2,STAT3 and p-STAT3 and the analysis of its association with astrocytoma grades were identified by immunohistochemical staining.PTP-MEG2 human protein expression plasmid and PTP-MEG2 specific sh RNA plasmid were constructed.The above plasmids were transiently transfected into the glioblastoma cell line U87 and the overexpression of PTP-MEG2 and the interference effect of PTP-MEG2 sh RNA were verified by q RT-PCR from the m RNA level.After establishing the cell model of bidirectional regulation of PTP-MEG2 expression,the expression of EGFR,p-EGFR,STAT3 and p-STAT3 protein after PTP-MEG2 overexpression and PTP-MEG2 interference were detected by Western blot.The effect of bidirectional regulation of PTP-MEG2 on proliferation of U87 cells was examined by MTT assay.Transwell assay was used to detect the effect of bidirectional regulation of PTP-MEG2 on U87 cell invasion.The effect of bidirectional regulation of PTP-MEG2 on the apoptosis of U87 cells was detected by flow cytometry.It was verified by GST-pull down and IP experiments whether PTP-MEG2 had direct binding to STAT3 in vitro and in vivo.Results:1.PTP-MEG2 can mediate dephosphorylation of EGFR and STAT3.2.PTP-MEG2 can further inhibit the proliferation and invasion of glioblastoma cells through the above-mentioned effects and promote its apoptosis.Specific interference with endogenous PTP-MEG2 can increase the phosphorylation level of EGFR,and at the same time enhance the phosphorylation level of STAT3,increase the proliferation and invasion of glioblastoma cells while down-regulate their apoptotic capacity.3.PTP-MEG2 and STAT3 can interact with each other both in vitro and in vivo,suggesting that STAT3 is a specific substrate for PTP-MEG2.4.The protein expression level of PTP-MEG2 was negatively correlated with the grade and poor prognosis of astrocytoma.Conclusion: PTP-MEG2 can mediated the dephosphorylation of both EGFR and STAT3,inhibiting the proliferation and invasion of astrocytoma cells.This finding is expected to provide a theoretical basis and new ideas for clinical treatment of astrocytoma and the related drugs development.