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Expression And Functional Analysis Of Tumorigenesis Asb11-1 And Cardiogenesis Xirp1

Posted on:2009-03-23Degree:MasterType:Thesis
Country:ChinaCandidate:J J HuFull Text:PDF
GTID:2194360245966487Subject:Genetics
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Tumor have been threatening and damaging human beings severely. In order to cure the tumor,it is a critical prerequisite to study the tumor-inducing genes and understand the mechanisms underlying the causes of this kind of diseases.As it known,mistakes in spatiotemporal expression of critical genes result in tumor.The identification of the molecular pathways that control tumorigenesis will provide a basic understanding of how tumor is taken place.In this study,we have cloned a novel member of human ankyrin repeat and SOCS box containing protein fmily(ASB),named as ASB11-1, from a human placental cDNA library.The gene encodes a protein containing six Ankyrin repeats at the N-terminus and one SOCS box at the C-terminus.ASB11-1 gene has seven exons separated by six introns and is mapped to human chromosome Xp22.31.RT-PCR is used for detecting the expression difference of ASB11-1 between cancer cells and normal tissue,find that the expression of gene is increased very significantly,suggesting that the gene may be associated with cancer. Northern blotting shows that the gene transcripts two different isoforms, one 2.9 kb and another 5.0 kb,which both are specially expressed in skeletal muscle and heart with defferent levels.In COS-7 cells,ASB11-1 proteins are localized to both the nucleus and the cytoplasm.In the analyses of p53 and p21 signaling pathways,overexpression of ASB11- 1 in MCF-7 cells activates the transcriptional activities of p53 and p21. RT-PCR and western blot show that overexpression of ASB11-1 upregulates the expression levels of p53 and p21.It has been reported that XIRP1 specially expressed in human heart and skeletal muscle,the orthologous gene of XIRP1 in chicken can be involved in BMP-Nkx2.5-MEF2C signaling way and participate in morphogenesis of heart.The expression level of XIRP1 is significantly reduced in Nkx2.5 and MEF2C knockout mice.This fact proves that Nkx2.5 and MEF2C can regulate the expression level of XIRP1.In order to understand how the promotor works,I clone a 2.1kb fragment of the 5' upstream seguence from the start code ATG of XIRP1.Then the 2.1kb fragment is deduced to some subfragments 1.9kb,1.2kb,1.0kb,0.8kb, 0.6kb,0.3kb and 0.2kb.These subfragments are cloned into pGL3-basic vector.Luciferase activity assay identified that the core promoter region lies in the site of-127-77 from the start code ATG.Point mutation assay finds that the core promoter activity decreased significantly,so it confirmed that the Nkx2.5,MEF2C,Hand2 site is real in this promoter. In this study we find the Hand2 site in this promotor is real for the first time.
Keywords/Search Tags:ASB11-1, p53, p21, Hand2, Nkx2.5, MEF2C, tumor, promoter
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