Study On The Molecular Mechanism Of SHP-1 Affecting Proliferation, Differentiation And Apoptosis Of Acute Promyelocytic Leukemia Cells Through PI3K/AKT Signaling Pathwa

Objective:The purposes of the research are to study the expression level of SHP-1 in APL patient tissues and cell line NB4,to investigate the effect of SHP-1 expression on APL cell proliferation,differentiation,and apoptosis in vivo and in vitro,as well as the effect of SHP-1 expression on PI3K/AKT signaling pathway and to explore its role in the occurrence and development of APL.Methods:1.The relative expression level of SHP-1 in bone marrow tissues of newly diagnosed APL patients and negative controls was detected by immunohistochemistry,and the expression level of SHP-1 in APL cell line NB4 and PBMCs from 3 normal volunteers was tested by Western Blot and RT-q PCR.2.APL cell line NB4 was infected with lentivirus,and overexpressed SHP-1 cell line NB4-OE and negative control cell line NB4-NC were constructed.The difference of SHP-1 expression in NB4-OE,NB4-NC,and untreated cell line was detected by Western Blot and RT-q PCR.3.The cell proliferation was detected by Ed U and CCK-8 assay,and the influence of SHP-1 on cell differentiation and apoptosis was tested by flow cytometry.4.Western Blot assay was performed to detect the influence of SHP-1 on the expression level of key proteins AKT,PI3 K,p-AKT,and p-PI3 K in PI3K/AKT signaling pathway of APL cell line NB4.5.The growth and proliferation of APL cells in nude mice were studied by constructing in vivo transplanted tumor model.6.All the experiments were performed three times,and the results were reported as mean ± SD.For data analysis,an unpaired two-tailed Student’s T-test was used to compare the differences between each group.Statistical computations were performed using Graph Pad Prism 8.0.A p value less than 0.05 was considered statistically significant.Results:1.Immunohistochemical method was used to detect the relative expression of SHP-1 in bone marrow tissues of APL patients,and Western Blot and RT-q PCR were used to detect the expression of SHP-1 in APL cell line: the expression of SHP-1 in bone marrow tissues of APL and APL cell line NB4 was down-regulated.2.RT-q PCR was used to detect the level of SHP-1 in each group after lentivirus transfection: the expression level of SHP-1 in the NB4-OE group was increased.3.CCK-8 and Ed U cell proliferation assay showed that the proliferation ability of NB4-OE group decreased.Cell differentiation and apoptosis by flow cytometry showed that cell differentiation and apoptosis were increased in NB4-OE group.4.The key protein level of PI3K/AKT signaling pathway was detected by Western Blot: The levels of PI3 K and AKT were not significantly changed in SHP-1overexpression group,while the levels of p-PI3 K and p-AKT were decreased.5.Transplanted tumor experiment in nude mice showed that the proliferation rate of transplanted tumor in NB4-OE group was decreased.Conclusion:As a tumor suppressor gene of APL,SHP-1 can inhibit the proliferation of APL cells and promote cell differentiation and apoptosis in vivo and vitro,the mechanism of SHP-1 may be related to the dephosphorylation of PI3K/AKT signaling pathway.