Preliminary Study On The Differentiation Function Of RREB1 In APL Cell Lines And Its Mechanism

PART ? DISCUSSION ON THE RELATIONSHIP BETWEEN RREB1 AND APL DIFFERENTIATIONObjective: To investigate whether RREB1 is closely related to the differentiation of APL cells.Methods: The mRNA expression of RREB1 in bone marrow and healthy adults of APL patients was analyzed by oncomine database.Quantitative PCR was performed to detect the expression level of RREB1 in myeloid leukemia cell lines.After 72 h treatment with 1 ?M ATRA on NB4 and hl-60 cells,changes in RREB1,CD11 b and CEBP? were detected by western blot.Indirect immunofluorescence was used to observe the expression and distribution of RREB1 in NB4 and hl-60 cells.Results: RREB1 was highly expressed in APL patients and APL cell lines,and ATRA inhibited the expression of RREB1 significantly.Western blot results showed that after ATRA treated APL cell lines for 72 h,CD11b and CEBP? protein levels increased,while RREB1 protein expression decreased significantly.Indirect immunofluorescence results showed that RREB1 was mainly distributed in the nucleus of NB4 and hl-60,and its protein expression was significantly down-regulated after ATRA treatment,but its localization was not affected.Conclusion: The expression of RREB1 in APL is significantly higher than that in normal people,and ATRA can significantly inhibit RREB1 expression in APL cell lines,all these indicate that RREB1 is likely to play a role in the differentiation process of APL.PART ? RREB1 BLOCKS THE DIFFERENTIATION OF APL CELL LINES BY REGULATING MIR-145 AND ERK/CEBP? PATHWAYSObjective: To investigate the effect of interference with RREB1 gene expression on the differentiation of acute promyelocytic leukemia cell lines(NB4,HL-60)and its mechanism.Methods: RREB1 gene was knocked down by lentivirus construction,and cell morphology and differentiation were detected by Swiss staining.Flow cytometry was used to detect the proportion of CD11 b positive cells.Expressions of RREB1,CD11 b,CEBP? and pERK were detected by western blot.Real-time fluorescence quantitative PCR was used to detect the expression of mir-145.RREB1 was overexpressed by plasmid transfection,and CD11 b expression in each group after ATRA treatment was detected by flow cytometry(results were not presented).Western blot analysis of the effects of pERK inhibitors and mir-145 inhibitors on the differentiation-related proteins of stable cell lines.Results: Down-regulation of RREB1 was successfully proven.Swiss staining showed enhanced differentiation of NB4 and HL-60 cells,showing a tendency of nucleus deviation and lobulation.Flow cytometry detected that the proportion of CD11 b positive cells after RREB1 knockdown was significantly higher than that of the negative control group.Western blot also detected upregulation of granulocyte differentiation marker proteins CD11 b and CEBP?,and upregulation of pERK.Real-time fluorescence quantitative PCR detected a significant increase in the expression of miR-145.Moreover,pERK inhibitors and miR-145 inhibitors could counteract the differentiation enhancement induced by RREB1 knockdown,suggesting that RREB1 plays a role in blocking cell differentiation in APL,and is related to the inhibition of RREB1 on mir-145 and pERK/CEBP? pathway.Conclusion: RREB1 affects the differentiation of APL cells through regulation of miR-145 and pERK/CEBP? pathways.