A Primary Study Of LRP16 Expression Regulation, Gene Function And Its Expression In Leukemia Cells

LRP16 is a novel gene which was cloned from human mononuclear cells by Yuli in 1999 using restriction length genomic scanning(RLGS) and the cDNA was isolated using the rapid amplification of cDNA end (RACE) technique (GeneBank Accession No. AF202922). The full length of LRP16 gene transcriptional copy has 1225 base pair of nucleotides and contains at least two open reading frames. The short coding protein is a nuclear protein, while the cell sub-location of the longer one is not clear.In order to explore the possible regulation mechanism of LRP16 gene expression, study the novel gene's function and its expression pattern in leukemia cells, the present study first cloned promoter and sub-clone promoter molecules, then constructed sub-LRP16 gene promoter-pGL3-Basic vectors. After that, Luciferase activities of all sub-clone promoter sequences were analyzed. At last, the possible cis-acting elements were analyzed by computer-aid system. Computer-aid neurological system was used to predict the primary and the second structure of LRP16 coding protein, as well as its putative evolutionary tree. In the experiment of laboratory part, we first construct a eukaryotic expression vector containing the long type ORF of LRP16 gene and its expression was detected in K562 cells. The effects of LRP16 over expression on cell proliferation and its cell cycle were observed as well.The LRP16 gene expression pattern was analyzed by the scanning of EST database, SAGE database, GeneNote database and 2DPAGE database. Based on these prediction results, RT-PCR method was used to detect the LRP16 expression pattern in leukemia cell lines and primary leukemia cells from patients.Results showed: ㎜RP16 promoter is a typical class II eukaryotic promoter with a TATA box and several GC boxes, which has a core positive regulation sequence from -600bp to -200bp and a few putative negative czs-acting elements locating from -2000bp to -lOOObp. LRP16 gene promoter has both general and special cis-acting elements relating to cell cycle, hematopoiesis, cell proliferation, carcinogenesis and acute reacting process. (DLRP16 may have three types of ORFs, the molecular weight of the longest ORF coding protein is 35505.1 ID and the theoretical pi value is 9.58. LRP16 coding protein is rich in Glycine (11.4%) and Leucine(ll.l%)> which has a complex secondary siruciur6 witrr 36.00% of a-helix, 13.85% of 8-sheet and 50.15% of other secondary structures. Long type LRP16 coding protein has one N-glycosylation site, six protein kinase C phosphorylation sites, five casein kinase II phosphorylation sites, two tyrosine kinase phosphorylation sites, three N-myristoylation sites and one amidation site locating in 18 different positions. The coding protein has a similarity domain with C terminal sequence of macroH2Al. LRP16 coding protein has a very conserved evolutionary domain in it sequence, which is expressed in nearly all kinds of species. The over expression of LRP16 long type ORF accelerates the proliferation of leukemia cell line K562 and this is partly due to shorten the time from GO phase to Gl phase and S phase. ㎜RP16 gene expressed nearly in all organs, tissues and cells, including bone marrow, spinal cord blood, spleen, liver, lungs, brain and heart et al, while it has no expression in human cerebrospinal fluid, human plasma and HepG2 secreted Proteins. All these showed that LRP16 coding protein is not a secreted protein. 甃RP16 gene coding protein may be a leukemia related protein, which may has somewhat function relation to leukemia drug resistance and relapse.