Differential Expression Of MS4A7 Gene In U-937 Differentiation And Identification Of Its Promoter

OBJECTIVE:To study the differential expression of MS4A7 gene in Acute Monocytic Leukemia cell line U937 during its differentiation. To explore the correlation of MS4A7 gene expression in different phase of leukemia, we have revealed the main signal pathways which controlled MS4A7 gene expression and found the relationship between leukemia and MS4A7 expression in the abnormal signal transduction. We analyzed MS4A7 promoter activity in leukemia and non-leukemia using a a MS4A7 promoter-derived luciferase reporter system and cloned 5 serial deletion constructs from MS4A7 promoter. We also observed the transcription factors which regulated the MS4A7 promoter activity.METHOD:1. To induce differentiation of U937, cells in logarithmic growth were treated with 50 ng/ml Phorbol 12-myristate 13-acetate (PMA) and collected total RNA at the time of Od, 1d,3d,5d. Total RNA were reverse to cDNA and detection MS4A7 gene expression through Real-Time PCR.2. To test the role of signal pathway in differentiation, U937 cells were pre-incubated for 6 h with 5μM of the MEK-1/-2 inhibitor U0126.50μM of p38 MAPK inhibitor SB230580 and JNK inhibiter SP600125. After pre-incubating of the inhibiters, one group were incubated with PMA for 48h, the other group were treated with nothing. Collected total RNA and reverse to cDNA and detection MS4A7 gene expression through Real-Time PCR.3. The promoter region of human MS4A7 gene was obtained from PCR amplification and was inserted into the luciferase reporter gene vector pGL3-Basic through restriction enzyme digestion to get pGL3-MS4A7-Promoterl-5 plasmids. To confirming the sequence, the insert was cut out by restriction enzyme digestion and sequence. The plasmid pGL3-MS4A7-Promoter 1-5 was transfected into leukemia(HL-60.U-937) and non-leukemia (HeLa,SKOV-3)cells and the luciferase activities were measured to know the activities of MS4A7 promoter. 4. To develop a real-time PCR-based chromatin immunoprecipitation (ChIP) assay for determining the effect of transcription factors ATF2 and ELK1 in the regulation of MS4A7 gene promoter.RESULTS:1. Treatment of U937 cells with PMA induces functional and morphological features that are characteristic of differentiated macrophages. We found that PMA had a remarkable enhancement of MS4A7 expression (p＜0.05). With prolonged stimulation of PMA, the expression level raised higher. After 3-5d, the gene expression were maintain constant (p<0.05).2. Compared with normal cultured cells, gene expression differences in the cells treated with U0126 and SP600125 was no statistically significant (P>0.05). When p38 kinase pathway was inhibited by sb203580, MS4A7 expression had a remarkable depression. Compared with differentiation cells induced by PMA, gene expression differences in the cells pre-treated with U0126 and SP600125 was no statistically significant (P>0.05). MS4A7 expression had a depression in the pre-incubated with SB23O580 (P＜0.05).3. After restriction enzyme digestion and sequence, lusiferrase reporter vector pGL3-MS4A7-Promoterl-5 containing human MS4A7 promoter can be constructed successfully. After transfection and fluorescence analysis, the MS4A7 promoters reflected different activities in leukemia and non-leukemia. The region between-1536bp～704bp had the strongest activities in leukemia cells.4. We did not detect the signal of MS4A7 promoter amplification after precipitating of anti-ATF2 antibody and anti-ELKl antibody.CONCLUSION:1. When U-937 cell were induced differentiation to macrophagocyte by PMA, MA4A7 gene expression had a remarkable raise. We inferred that MS4A7 gene play an important role in cell differentiation.2. There are some relationship between MS4A7 gene expression and p38 MAPK pathway activation. Considering the role of p38 MAPK in differentiation of hematopoietic cells, we think p38 participate the U937 cell differentiation induced by PMA, which is from immature cells to macrophagocyte. p38 MAPK may active some transcription factors downstream which may promote MS4A7 gene expression.3. The MS4A7 promoters reflected different activities in leukemia and non-leukemia. In leukemia cell lines, the region in-703bp～79bp has weaker promotion activity.The region in-1536bp～704bp has a strong activity,the region may play a important role in MS4A7 gene transcription.4. There has not been shown that MS4A7 promoter has ATF2 and ELK1 binding site. We need further research to know about the transcription factor regulating MS4A7 gene expression.