Effect Of SHP-2 Activating Mutation On Proliferation And Invasion Of Glioma Cells

Objective:Glioma is the most common primary tumor in central nervous system,about 80%of intracranial tumors is glioma.Glioma is aggressive with strong proliferation,invasion and migration ability.After standard treatment,the median survival is less than18 months and the 5-year survival ratio is less than 5%.Therefore,it has become a clinical focus of attention to find new therapeutic methods or targets for glioma.SH₂domain-containing protein tyrosine phosphatase-2（SHP-2）is one of the members of protein tyrosine kinase family,SHP-2 is widely expressed in human tissues,organs and cells,participating in embryonic development and regulating cell proliferation,differentiation,adhesion,migration and other biological behaviors.However,the correlation between SHP-2 and glioma is unclear,so the present study aims to explore the SHP-2 activating mutation in glioma and its relationship with clinicopathological features.The present study will explore SHP-2^D61Gmutation promoting malignant biological behaviors of glioma cell and mechanism.We next furtherly explore the function of SHP-2^D61Gmutation by xenograft model of mice.Methods:Case selection:45 glioma patients in the First affiliated hospital of Xinjiang Medical University and the Fourth affiliated hospital of Xinjiang Medical University from September,2015 to June,2016were selected as research subjects.All patients were diagnosed by pathology,15 patients diagnosed as cerebral hemorrhage were assigned as control.All samples were granted by ethics committee of hospital.Inclusion criteria:（1）all 45 glioma patients were first admission,no radiotherapy and chemotherapy before surgery;（2）good quality of paraffin section;（3）complete clinical and pathological data;（4）all tumors were diagnosed as glioma after surgery.SHP-2^D61G mutationdetection:DNA of tissues were isolated,then the mutation site was detected by droplet digital PCR（ddPCR）.The difference of SHP-2^D61G61G mutation between glioma and control was evaluated by comparison;The difference of SHP-2^D61G mutation among glioma with different stages was evaluated by comparison;The correlation between SHP-2^D61G mutation and clinicopathological features was performed by correlation analysis.Human brain glioma U251 cellline was purchased,then transfected with pcDNA3.1 empty vector and pcDNA3.1 SHP-2^D61G respectively.Establishment of U251 cell transfection and stable cell line:G418 with different concentration were placed into the cell,the optimal screening concentration of G418 was determined by selecting the lowest working concentration which could cause cell death.The expression of SHP-2 after transfection and screening was performed by western blot.Cell viability of U251 was detected by MTT assay.Colony forming ability was investigated by colony formation assay.Apoptosis of U251 cell line was explored by flow cytometry.Invasion and migration ability of U251 cell lines were completed by transwell chamber assay.The expression of ERK1/2,AKT and p38 and their phosphorylation were detected by western blot.U251 cells after transfection were treated with p38 MAPK pathway inhibitor（SB203580）,MEK pathway inhibitor（U0126）and PI3K-AKT pathway inhibitor（PF-04691502）respectively.Then the expression of ERK1/2,AKT and p38 and their phosphorylation were semi-quantified by western blot.Lentivirus plasmids of SHP-2^D61Gmutation and empty vector were constructed.Then the vector were packaged by lentivirus and transfected into U251 cell lines.After transfection,cell lines stably expressed SHP-2SHP-2^D61Gmutation and cell lines transfected with empty vector were screened by puromycin.The U251 cells transfected with plasmids were subcutaneously inoculated into C57BL/6J mice,U251 cells without transfection were subcutaneously inoculated into C57BL/6J mice and set as control.Weight changes of mice were recorded and the curve of weight change was completed,the final tumor sizes of mice were evaluated by whole-body optical imaging system.Death time of mice was recorded and the survival curve was completed by SPSS software.Results:SHP-2^D61G abundance of control and glioma were o and 3.36±1.08（%）respectively.SHP-2^D61Gmutation related with stages of glioma.The higher the grade is,the higher abundance of SHP-2^D61Gwill be.SHP-2^D61Gmutationdidn’t relate with gender,age and tumor location,but relate with tumor size.Comparing with control,the expression of SHP-2 in glioma was elevated,besides,the expression of SHP-2 in SHP-2^D61Gmutation positive glioma was higher than that in SHP-2^D61Gmutation negative glioma.Comparing with control group,the expression of SHP-2 was elevated in cells transfected with SHP-2^D61Gplasmid.SHP-2^D61Gmutation promoted the cell viability of U251.SHP-2^D61Gmutation increased the colony forming ability.SHP-2^D61Gmutation decreased apoptosis of U251 cell.SHP-2^D61Gmutation promoted invasion and migration ability of U251 cell.SHP-2^D61Gmutation elevated the expression of p38,ERK,AKT and their phosphorylation.The expression of p38,ERK,AKT and their phosphorylation was decreased after inhibitor treatment.Comparing with control group,there was no statistical difference among groups in 5 days after inoculation,however,a significant increase in SHP-2^D61Gmutation group was observed since day 6after inoculation（P<0.05）.Survival analysis showed that compared with control group,the survive time of mice in SHP-2^D61Gmutation group was obviously decreased（P<0.01）.Conclusion:Normalbraintissuedoesn’thaveSHP-2^D61Gmutation,but SHP-2^D61Gmutation was observed in glioma tissues.The SHP-2^D61Gmutation in glioma relates with tumor size and tumor grade.The expression of SHP-2 was elevated in glioma,and SHP-2^D61Gmutation promotes the activation of SHP-2.SHP-2^D61Gmutation promotes malignant biological behaviors of glioma cells,including cell apoptosis,proliferation,invasion,migration and clone.SHP-2^D61Gmutation may promote malignant biological behaviors of glioma cells via activating p38,ERK1/2 and AKT signaling pathway.SHP-2^D61Gmutation may become a potential target for glioma treatment.SHP-2^D61Gmutation promotes proliferation and growth of U251 cells.SHP-2^D61Gmutation in glioma patients may shorten their survival time.