Exploring The Role And Mechanism Of PHLPP2 Overexpression In Burkitt Lymphoma

Burkitt lymphoma(BL)is a highly invasive non-Hodgkin lymphoma from B-cell.High-intensive chemotherapy can be very effective in the treatment of BL,especially for younger patients.However,the treatment effect for the elderly patients and immunodeficient patients is poor,and it puts emphasis on the need of alternative therapy.PHLPP2 is a negative regulator of PI3K/AKT pathway and can directly dephosphorylate the Ser473 site of AKT.At present,studies have shown that PHLPP2 is abnormally expressed in a variety of solid tumors.Up-regulating the expression of PHLPP2 plays the role of tumor suppressor factor,inhibiting the proliferation of tumor cells,blocking the cell cycle progression and inducing the apoptosis of cells.In order to provide a new theoretical basis for clinical treatment of Burkitt’s lymphoma,this subject explored the biological function of PHLPP2 gene in Burkitt’s lymphoma and found the objective of targeted therapy for Burkitt lymphoma from a new perspective.The research of this topic is divided into the following two parts:Part Ⅰ The Construction of Burkitt Lymphoma Cell Lines with Stable Overexpression of PHLPP2.Objective: Constructing the lentiviral vector with PHLPP2 overexpression and screening CA46 cell lines and RAJI cell lines with PHLPP2 overexpression.Methods: 1.PHLPP2 c DNA was obtained by PCR,ligated to vector,and verified by sequence testing.2.Lentivirus was packaged and concentrated,and the titer of lentivirus was detected by flow cytometry.3.CA46 cells and RAJI cells were infected by lentivirus.The infection efficiency was observed under fluorescence microscope and screened by puromycin.The expression level of PHLPP2 was detected by RT-PCR and Western blot.Results: 1.The lentiviral vector with PHLPP2 overexpression was successfully constructed and verified by sequence testing.2.CA46 cell lines and RAJI cell lines with PHLPP2 overexpression were successfully constructed and verified by RT-PCR and Western blot.Conclusion: Burkitt lymphoma cell lines with PHLPP2 overexpression can be successfully constructed.Part Ⅱ The Effect of PHLPP2 Overexpression on the Proliferation,Cell Cycle and Apoptosis of Burkitt lymphoma Cell Lines and its Mechanism.Objective: Exploring the effects of PHLPP2 overexpression on proliferation,cell cycle and apoptosis of Burkitt lymphoma cell lines and its mechanism.Methods: 1.The effect of PHLPP2 overexpression on the proliferation of CA46 cells and RAJI cells was detected by CCK-8 method.2.The effect of PHLPP2 overexpression on cell cycle of CA46 cells and RAJI cells was detected by flow cytometry.The related proteins of cell cycle was detected by Western blot.3.The effect of PHLPP2 overexpression on apoptosis of CA46 cells and RAJI cells was detected by Hoechst33342 and PI staining method.The related proteins of apoptosis was detected by Western blot.4.The changes of PI3K/AKT pathway was detected by Western blot.Results: 1.Overexpression of PHLPP2 inhibited the proliferation of CA46 cells and RAJI cells.2.Overexpression of PHLPP2 blocked the cell cycle progression of CA46 cells and RAJI cells,up-regulated the expression of P21 and down-regulated the expression of Cdk4 and Cycling D3.3.Overexpression of PHLPP2 promoted the apoptosis of CA46 cells and RAJI cells,down-regulated the expression of PARP and Bcl-2,and up-regulated the expression of cleaved PARP and Bax.4.Overexpression of PHLPP2 down-regulated the expression of p-AKT(Ser473)in CA46 cells and RAJI cells.Conclusion: PHLPP2 overexpression suppresses PI3K/AKT pathway by blocking the phosphorylation of ATK(Ser473),inhibits cell proliferation,blocks the progress of cell cycle and induces the apoptosis in Burkitt lymphoma cells.Bcl-2 family and Caspase family are involved in the regulation of cells apoptosis.