Expression profiling of HMGB1 suppressed MCF-7 cells

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Expression profiling of HMGB1 suppressed MCF-7 cells

 

Author: Peng, Xiao
Title: Expression profiling of HMGB1 suppressed MCF-7 cells
Degree: Ph.D.
Year: 2007
Subject: Hong Kong Polytechnic University -- Dissertations.
Protein binding.
Eukaryotic cells.
Gene expression.
Cell proliferation.
Department: Dept. of Applied Biology and Chemical Technology
Pages: xix, 182 leaves : ill. (some col.) ; 30 cm.
Language: English
InnoPac Record: http://library.polyu.edu.hk/record=b2116756
URI: http://theses.lib.polyu.edu.hk/handle/200/1973
Abstract: The high mobility group protein HMGB1 is the most abundant non-histone chromosomal protein in mammals and has counterparts in all eukaryotes. It has long been known as an architectural transcription factor involved in gene activation as well as in other nuclear processes. More recently, HMGB1 has been found to have extra-cellular functions as a late mediator in immune response and also as a cytokine. We have been interested in how the human HMGB1 gene is regulated as well as how HMGB1 regulates other genes in the cell. In this study, an anti-sense strategy was used to suppress the HMGB1 expression level in the human breast cancer MCF-7 cell line. An MCF-7 cell line that expresses HMGB1 at only half the level of the original cell line was established. The expression profiles of these two cell lines were compared using cDNA microarray and the genes differentially expressed at a significant level were identified in these two cell lines. Among these genes, 96 were down regulated and 76 were up regulated in the HMGB1 suppressed MCF-7 cells. Real-time RT-PCR was also used to check the expression levels of some of the differentially expressed genes. Furthermore, these genes were classified into 11 functional groups including transcription factors, cell cycle related factors, apoptosis regulators, kinases and metabolism-related proteins, etc. The potential regulatory roles of HMGB1 on some of these groups were then discussed. In addition, to find out the possible interactions among these differentially expressed genes, Pathwayassist Software was used for analysis. It was found that the products of 16 of these differentially expressed genes fall into a complex network with p53 as the key node. The expression levels of the p53 and MDM2 genes were found to be significantly lower in the HMGB1 down-regulated MCF-7 cells. This indicates the importance of HMGB1 in p53 expression and cell cycle checkpoint control. Based on the possible role of HMGB1 in the p53 network, investigattion on whether HMGB1 could regulate some potential downstream genes at the transcriptional level was carried out. An HMGB1 expression plasmid together with the p53, the MDM2 or the E2F1 promoter-containing reporter plasmid were co-transfected into the MCF-7, the HMGB1 suppressed MCF-7 and the p53-null human osteosarcoma Saos-2 cell lines to examine the transcriptional effects of exogenous HMGB1 on these promoters. In a parallel experiment, a p53 expression plasmid was also included to find out if the p53 protein may influence the regulatory effect of HMGB1 on these promoters. The luciferase assay results demonstrated that HMGB1 does not regulate the p53 promoter directly but may affect the expression of p53 via its regulation on the MDM2 and the E2F1 promoters. The action of HMGB1 on the MDM2 promoter is in a p53-dependent manner whereas its activation of the E2F1 promoter is p53-independent. Therefore, HMGB1 is an important regulatory factor in the p53-MDM2-E2Fl network.

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