Effect Of Toxoplasma Gondii ROP16 Protein Of Human Acute Myeloid Leukemia HL60 Cells And Mechanism

ObjectiveToxoplasma gondii ROP16_Ⅰ/Ⅱ/Ⅲprotein was over-expressed in HL60 cells by infecting with lentivirus to investigate the changes of HL60 cell（acute myeloid leukemia cell）on biological function（proliferation,apoptosis,cycle,migration and invasion）,and the preliminary mechanisms related to JAK-STAT pathway and the differences in the effects of ROP16 polymorphism on HL60 cells,so as to provide new ideas for the clinical treatment of acute myeloid leukemia and the anti-tumor research of parasite secreted proteins.Method1.Using lentivirus transfection technology,HL60 cells was infected with the lentivirus,then observed the green fluorescence under the fluorescence microscope;Real time PCR and Western-blot was use to detect the ROP16 mRNA and protein respectively.2.After lentivirus successfully transfected HL60 and achieved high transfection rate,Flow cytometric was used to detect the apoptosis rate of cells and the cell cycle,CCK-8 was used to detect cell proliferation,The influence of ROP16 on the invasion and migration of HL60 was detected by Transwell experiment,Real time PCR and Western-blot was used to detect the expression of Bax and Bcl-2 mRNA and protein respectively.3.PCR ARRAY was used to screen the up-regulated and down regulated genes related to JAK-STAT signaling pathway,and to explore the possible mechanism.Real time PCR and Western blot were used to verify the results.Result1.Three different types of ROP16 protein lentiviral vectors was successfully constructed and transfected into HL60 cell line.It was obvious that strong green fluorescence under the fluorescence microscope.Real time PCR and Western-blot showed that the expression of ROP16 mRNA and protein increased in the HL60 cells,and observed obvious 96KDa size Bands.2.Flow cytometry showed that there was neither obvious apoptosis in the five groups of cells nor significant difference.Compared with the HL60 group,the proportion of cells in the G2 phase of HL60-ROP16Ⅰ_/Ⅲcells was significantly reduced;CCK8 showed that there was no significant difference on proliferation between the experimental groups.Transwell experiment showed that the migration and invasion ability of HL60-ROP16_Ⅰ/Ⅱ/Ⅲcells was significantly lower than HL60 and HL60-Venus.Real time PCR and Western-blot showed that there was no significant difference in the expression levels of Bax and Bcl-2 mRNA and protein in the five groups of cells.3.The PCR ARRAY showed that compared with the HL60 group,HL60-ROP16_Ⅰ/Ⅱ/Ⅲgroups had at least 17 genes up-regulated,5 genes down-regulated,and 12 gene expression levels were significantly different,Real time PCR and Western-blot showed that HL60-ROP16_Ⅰ/Ⅱ/Ⅲgroups the protein expression levels of STAT3,STAT6,p-STAT3,and p-STAT6 were significantly higher than the other two groups.Conclusion1.Successfully constructed type I/II/III rop16 gene over-expression lentiviral vector,and and transfected into HL60 cells.2.In vitro experiments,the Toxoplasma gondii ROP16 protein can’t inhibit the activity of HL60 cells by directly affecting the proliferation,apoptosis and cycle.3.In vitro experiments,the Toxoplasma gondii ROP16 protein can reduce the migration and invasion ability of HL60 cells,among which typeⅡis the most obvious.4.Toxoplasma gondii ROP16 protein may effect the migration and invasion of HL60cells by regulating the JAK-STAT pathway,and there are significant differences in the expression levels of some genes among HL60-ROP16_Ⅰ/Ⅱ/Ⅲ groups.