Cynthia Ricini Rdna Transcription Repeat Unit Sequencing And Transcribed Spacer Function Analysis Of Liver Cancer-related Genes Of Lpts The, Hcrp1 And Lis1 The Transcriptional Regulation Research

Suiquan Wang (Biochemistry and Molecular Biology) Directed by Prof. Mujun Zhao and Prof. Tsaiping Li The thesis consists of two parts. The first part is "Complete Sequence of theSilkworm Attacus ricini rDNA Repeat, Determination of the TranscriptionalInitiation Site and Functional Analysis of the Intergenic Spacer" We subclonedthe rDNA repeat unit of silkworm Attacus ricini and determined its complete 10.3kbsequence. We also identified the transcriptional initiation site and a possibleprocessing site of pre-rRNA by primer extension method. Further sequence analysisrevealed that although nucleotide sequences of rDNA intergenic spacer (IGS) indifferent species showed little homology, certain conservation of IGS in thehigher conformation level exists between Attacus ricini and other eukaryoticorganisms by. IGS region of Attacus ricini is only 2670bp long, much shorter thanother higher eukaryotic organisms, but it contains all the components necessaryfor eukaryotic polymerase I transcription regulation. So we can use this silkwormas an important model to study the rRNA transcription regulation. The second part is "Identification and functional analysis of the promotersfrom three liver cancer related genes including LPTS, HCRP1 and Lis1". The genefor LPTS encodes a protein that is a well-characterized telomerase inhibitor.HCRP1, which is identified by our lab, is a new gene located in chromosome 8p22.Both of the LPTS and HCRP1 are downregulated in liver cancer cells and tissues.Lis1 is an important gene participating in the mitosis and migration of neuronsand it was found to be upregulated in liver cancer by our lab. In this part, thepromoters of these three genes were isolated and analyzed for their transcription iiactivities. Transcription initiation site and four DNase I hypersensitive sitesin the LPTS promoter region were also identified. Results showed that LPTS promoteractivity had no significant difference in cultivated liver cells while sensitivityof promoter region to DNase I had diversities between LPTS differently expressedliver cells suggesting that transcription regulation in chromatin level mayfunction in LPTS expression. The promoter methylation status was also examinedin these three genes. The results showed that no promoter hypermethylation foundin either gene, indicating that promoter hypermethylation isn't the factorcausing their downregulation in HCC. LOH analysis of HCRP1 and LIS1 were alsocarried out in order to elucidate the mechanisms that cause these genes' aberrantexpression in liver cancer. The frequency of LOH of LPTS and HCRP1 genes arerelatively high (34.5%-50%) in HCC, while no allelic imbalance of Lis1 gene wasfound in HCC. We suppose that LOH may be the main reason for the downregulationof LPTS and HCRP1. We also proved that reducing the endogenous Lis1 expressionby RNAi technology could inhibit the growth and clone formation ability ofcultivated HCC cells significantly suggesting the important roles of Lis1 genein the development of HCC. Further studying the expression regulation of livercancer related genes is important to elucidate the etiological and pathologicmechanisms of tumor development.