Transcription factor SOX-5 may enhances nasopharyngeal carcinoma progression by downregulating SPARC gene expression

鼻咽癌在東南亞一帶，是一常見的疾病，其致病的原因相當複雜，至今仍未能完全明瞭。本論文研究的主要目的，就是想找出與鼻咽癌早期致病有關的主要基因，研究這些基因在好發初期之鼻咽癌中的改變。我們用微陣列分析 (Microarray Analysis) 的方法，比較鼻咽癌細胞株以及鼻腔內膜上表皮細胞的初次培養 (Primary Culture)，兩者之間基因表現的差異。結果選出了十一個在兩者之間有差異表現的基因。再經由定量聚合酶連鎖反應 (Quantitative RT-PCR) 以及西方吸漬法 (Western Blotting) 的確認分析，發現 SPARC 基因在鼻咽癌細胞中的表現量顯著減少。進一步研究 SPARC 基因表現量減少的分子機制時，我們發現轉錄因子 (Transcription Factor) SOX-5 在鼻咽癌細胞中的表現量增加。如果用 shRNA抑制掉鼻咽癌細胞中 SOX-5 的表現，那麼 SPARC 基因的表現量就會增加。染色質免疫沉澱分析 (Chromosome Immunoprecipitation Assay, ChIP) 的結果，更證明 SOX-5 可以直接結合到 SPARC 基因的促進序列 (Promoter Region)，並抑制 SPARC 基因的表現。我們也發現，抑制掉鼻咽癌細胞中的 SOX-5 之後，鼻咽癌細胞的生長速率以及爬行速度均減緩；同樣的生理現象也發生在大量表現 SPARC 基因的鼻咽癌細胞之中。如果我們在抑制掉 SOX-5 的鼻咽癌細胞培養液中，加入抑制 SPARC 的抗體，則可以反轉抑制掉 SOX-5 所造成的生理現象。分析了六十六個鼻咽癌病人的檢體之後，我們發現 SOX-5 表現越多的病人檢體，該病人的預後也越差。根據我們的實驗結果，我們認為 SOX-5 可透過抑制 SPARC 基因的表現來影響鼻咽癌病程的進展，並且 SOX-5 很有可能是鼻咽癌的標的基因。Nasopharyngeal carcinoma (NPC) is prevalent in southeastern Asia, and its tumorigenesis is rather complex. The purpose of this research was to identify the pivotal genes that may be altered during the early stage of NPC progression. Eleven genes were selected by comparative microarray analysis of NPC versus normal nasomucosal cells. The expression of SPARC (secreted protein, acidic, cysteine-rich) was statistically significantly downregulated in NPC cells. In exploring the mechanism underlying the decreased transcription of SPARC in NPC cells, we found that the transcription factor SRY (sex-determining region Y)-box 5 (SOX-5) is upregulated in NPC cells. RNA interference of SOX-5 by short hairpin RNA (shRNA) in NPC cells caused a dramatic increase in SPARC and chromosome immunoprecipitation assay showed SOX-5 can bind directly to SPARC promoter, suggesting that SOX-5 acts as a key transcriptional repressor of SPARC. We further demonstrate that shRNA knockdown of SOX-5 suppressed the proliferation of NPC cells, as well as their migratory ability, which was also observed when SPARC was overexpressed in NPC cells. Alternatively, blocking SPARC with an antagonistic antibody reversed the effects of SOX-5 knockdown. In 66 NPC patients’ biopsy specimens, overexpression of SOX-5 in tumor cells correlated clinically with poor survival. Our study suggests that SOX-5 transcriptionally downregulates SPARC expression and plays an important role in regulation of NPC progression. SOX-5 is a potential tumor marker for advanced NPC prognosis.中文摘要 ………………………………………………………………………… 1bstract ………………………………………………………………………… 2hapter 1. Introduction ………………………………………………………… 3hapter 2. Materials and Methods ………………………………………………13hapter 3. Results ……………………………………………………………… 21hapter 4. Discussion …………………………………………………………… 27eferences …………………………………………………………………… 34ables ………………………………………………………………………… 46igures ………………………………………………………………………… 5

Transcription Factor Sox-5 Enhances Nasopharyngeal Carcinoma Progression by down-Regulating Sparc Gene Expression

Nasopharyngeal carcinoma (NPC) is prevalent in south-eastern Asia, and its tumourigenesis is rather complex. The purpose of this research was to identify the pivotal genes that may be altered during the early stage of NPC progression. Eleven genes were selected by comparative microarray analysis of NPC versus normal nasomucosal cells. The expression of SPARC ( secreted protein, acidic, cysteine-rich ) was statistically significantly down-regulated in NPC cells. In exploring the mechanism underlying the decreased transcription of SPARC in NPC cells, we found that the transcription factor SRY (sex-determining region Y)-box 5 ( SOX-5) is up- regulated in NPC cells. RNA interference of SOX -5 by short hairpin RNA ( shRNA) in NPC cells caused a dramatic increase in SPARC and chromosome immunoprecipitation assay showed that SOX-5 can bind directly to the SPARC promoter, suggesting that SOX-5 acts as a key transcriptional repressor of SPARC. We further demonstrated that shRNA knockdown of SOX-5 suppressed the proliferation of NPC cells, as well as their migratory ability, which was also observed when SPARC was over- expressed in NPC cells. Alternatively, blocking SPARC with an antagonistic antibody reversed the effects of SOX-5 knockdown. In 66 NPC patients, over-expression of SOX-5 in tumour cells correlated clinically with poor survival. Our study suggests that SOX-5 transcriptionally down-regulates SPARC expression and plays an important role in the regulation of NPC progression. SOX-5 is a potential tumour marker for poor NPC prognosis

Tumorigenesis and Neoplastic Progression NOLC1, an Enhancer of Nasopharyngeal Carcinoma Progression, Is Essential for TP53 to Regulate MDM2 Expression

Nasopharyngeal carcinoma (NPC) is one of the most common cancers among Chinese living in South China , Singapore , and Taiwan. At present , its etiological factors are not well defined. To identify which genetic alterations might be involved in NPC pathogenesis , we identified genes that were differentially expressed in NPC cell lines and normal nasomucosal cells using subtractive hybridization and microarray analysis. Most NPC cell lines and biopsy specimens were found to have higher expression levels of the gene encoding nucleolar and coiled-body phosphoprotein 1 (NOLC1) as compared with normal cells. Severe combined immunodeficiency mice bearing NPC xenografts derived from NOLC1-short hairpin-RNA-transfected animals were found to have 82% lower levels of tumor growth than control mice as well as marked tumor cell apoptosis. Measuring the expression levels of genes related to cell growth, apoptosis , and angiogenesis , we found that the MDM2 gene was down-regulated in the transfectants. Both co-transfection and chromatin immunoprecipitation experiments showed that tumor protein 53-regulated expression of the MDM2 gene requires co-activation of NOLC1. These findings suggest that NOLC1 plays a role in the regulation of tumorigenesis of NPC and demonstrate that both NOLC1 and tumor protein 53 work together synergistically to activate the MDM2 promoter i

NOLC1, an Enhancer of Nasopharyngeal Carcinoma Progression, Is Essential for TP53 to Regulate MDM2 Expression

Nasopharyngeal carcinoma (NPC) is one of the most common cancers among Chinese living in South China, Singapore, and Taiwan. At present, its etiological factors are not well defined. To identify which genetic alterations might be involved in NPC pathogenesis, we identified genes that were differentially expressed in NPC cell lines and normal nasomucosal cells using subtractive hybridization and microarray analysis. Most NPC cell lines and biopsy specimens were found to have higher expression levels of the gene encoding nucleolar and coiled-body phosphoprotein 1 (NOLC1) as compared with normal cells. Severe combined immunodeficiency mice bearing NPC xenografts derived from NOLC1-short hairpin-RNA-transfected animals were found to have 82% lower levels of tumor growth than control mice as well as marked tumor cell apoptosis. Measuring the expression levels of genes related to cell growth, apoptosis, and angiogenesis, we found that the MDM2 gene was down-regulated in the transfectants. Both co-transfection and chromatin immunoprecipitation experiments showed that tumor protein 53-regulated expression of the MDM2 gene requires co-activation of NOLC1. These findings suggest that NOLC1 plays a role in the regulation of tumorigenesis of NPC and demonstrate that both NOLC1 and tumor protein 53 work together synergistically to activate the MDM2 promoter in NPC cells