The Expression Of Differentiation-related Gene14 In Differentiation Of Leukemia Cell K562 And Cloning Of The Alternative Splicing MRNA

Leukemia is one of the malignancy of the hemopoietic organs,which is often originated from certain phase of differentiation of hematopoietic cells and show disorders of differentiation. Induction of differentiation of leukemia cells is an effective therapy and is used widely in administration of leukemia patients. Induction of differentiation is also a good method to study the mechanism of leukemia cell differentiation. Some researches showed that different leukemia cells can be induced differentiating along different lineages by different reagents. However, the mechanisms leading to differentiation are still not clear and need further works.Dif14 gene is a novel gene cloned and named by our laboratory in 2000. Dif14 gene was cloned from HMBA (hexamethylene bisacetamide)-induced leukemia cell line K562 which was differentiated along macrophage-like lineage. It suggests a potential association of dif14 with the differentiation of leukemia cells. To our knowledge, there have been no reports on the function of dif14 gene. In the previous studies, we cloned the cDNA sequence of dif14 gene long form, and it was constructed into the pGEM-7zf(+) vector.Objectives The present study was to investigate the expression of dif14 gene in differentiated myelogenous leukemia cells induced by HMBA and in different human normal tissues, we also aimed to clone its alternative splicing mRNA, so as to lay foundation for further understanding the function of dif14 gene.Methods1. Analysis of dif14 gene by bioinformatics tools.2. Dif14 mRNA was detected by Northern blot in K562 cells differentiation model induced by HMBA and in human different normal tissues.3. Construction of prokaryotic expression vector, expression and purification of DIF14 N-terminal protein in E. coli were done. We developed polyclonal antibody of DIF14 by immunization of rabbit. The expression of DIF14 protein in K562 cell differentiation model was investigated by using Western blot.4. Alternative splicing mRNA of dif14 gene was cloned by RT-PCR.Results1. Dif14 gene codes a putative nine-span transmembrane protein, the amino acid sequence of DIF14 protein was conserved in different species. We hypothesized DIF14 as an endocytosis receptor. 2. Both dif14 mRNA and protein expression were upregulated in induced K562 cells. The mRNA of dif14 was abundant in human normal skeletal muscles, kidney and liver tissues.3. Two transcripts of dif14 gene were found, they are 4.6kb and 2.5kb respectively. One of the transcripts was cloned and the alternative splice caused the deletion of 6th and 7th transmembrane regions of DIF14 protein.Conclusions1. Upregulation of both dif14 mRNA and protein level in HMBA-induced K562 cell model revealed that dif14 expression was associated with macrophage lineage differentiation. This is consistent with the hypothesis that DIF14 is a putative nine-span transmembrane endocytosis receptor.2. Dif14 gene has two alternative splice transcripts. One codes DIF14 full length protein, the other codes a protein with a deletion of 6th and 7th transmembrane regions of DIF14 protein.