O presente estudo teve o objetivo de verificar o padrão de expressão dos genes HOXB13 e HHEX em carcinomas epidermóides de boca (CEB) através das técnicas de Transcriptase Reversa em Reação de Cadeia Polimerase (RT- PCR) e Hibridização In situ (ISH). Fragmentos de tecido tumoral e de tecido não tumoral adjacente à lesão foram obtidos de 30 pacientes portadores de CEB no Serviço de Cirurgia de Cabeça e Pescoço do HC - FMUSP. As amostras tiveram seus cDNAs extraí dos e submetidos à amplificação por PCR. Os “ amplicons” foram visualizados sob luz UV por eletroforese em gel de agarose a 1% contendo brometo de etí dio. A amplificação dos genes foram correlacionadas com a classificação UICC, TNM, graduação histológica, localização e espessura tumoral, invasão de tecidos adjacentes, perineural e vascular. Após seqüenciamento dos “ amplicons” e confirmação dos genes foram confecionadas as sondas de mRNA para realização da técnica de hibridização in situ. Os resultados obtidos através da técnica de RT-PCR mostraram que: a amplificação dos transcritos dos genes HOXB13 e HHEX podem ser detectados tanto no carcinoma epidermóide quanto no tecido não tumoral adjacente à lesão; não existindo diferença na amplificação dos transcritos de ambos genes para os dois grupos de tecido estudados; a amplificação do transcrito do gene HOXB13 mostrou relação estatí stica com os fatores prognósticos: espessura tumoral, invasão perineural e invasão vascular; a amplificação do transcrito do gene HHEX mostrou relação estatí stica com os fatores prognósticos: invasão vascular,envolvimento com os tecidos adjacentes e uma relação inversa com a idade do paciente. A expressão dos transcritos dos genes HOXB13 e HHEX, detectados pela técnica de ISH, mostrou um padrão de marcação consistente e invariável para os tecidos analisados, estando expressos tanto em carcinoma epidermóide quanto no tecido não tumoral adjacentes à lesão. Os resultados apontam para uma correlação entre a expressão do HHEX e do HOXB13 e alguns fatores prognósticos importantes podendo representar um indicador prognóstico valoroso para o entendimento do comportamento biológico do CEB. / The aim of this study was to verify the HOXB13 and HHEX genes expression in oral squamous cell carcinoma (OSCC) using Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) and in situ Hybridization techniques. Tumoral tissues and adjacent non-tumoral oral mucosa specimens were obtained from 30 patients with OSCC at the Head and Neck Surgery Service HC (FMUSP). The samples were cDNA extracted and submitted to the RT-PCR technique. The amplicons were visualized in electrophoresis on a 1% agarose gel with ethidium bromide. Genes expressions were correlated with UICC staging, TNM stage, tumor location, tumor thickness, adjacent tissues involvement, vascular and perineural invasion, and cellular differentiation. Finally, direct sequence analysis was performed on PCR products to confirm cDNA sequence. Riboprobes were confectioned for in situ hybridization analysis. RT-PCR results showed HOXB13 and HHEX transcripts in both tumoral and non-tumoral tissue samples; no statistical correlation was verified between HOXB13/HHEX expressions and tumoral or non-tumoral tissues; there was a positive correlation between HOXB13 tumoral expression and tumor thickness, neural invasion, and vascular invasion; there was a positive correlation between HHEX tumoral expression and adjacent tissue involvement, vascular invasion, and a inverse relationship with patients age; ISH technique exhibited a consistent and invariable pattern of expression for both genes on both tumoral and non-tumoral tissue samples. Present results points out to a correlation between HOXB13 and HHEX expressions, and some important prognostic indicators, and this may represent a valuable tool to understand the biological behavior of OSCC.

O presente estudo teve o objetivo de verificar o padrão de expressão dos genes HOXB13 e HHEX em carcinomas epidermóides de boca (CEB) através das técnicas de Transcriptase Reversa em Reação de Cadeia Polimerase (RT- PCR) e Hibridização In situ (ISH). Fragmentos de tecido tumoral e de tecido não tumoral adjacente à lesão foram obtidos de 30 pacientes portadores de CEB no Serviço de Cirurgia de Cabeça e Pescoço do HC - FMUSP. As amostras tiveram seus cDNAs extraí dos e submetidos à amplificação por PCR. Os “ amplicons" foram visualizados sob luz UV por eletroforese em gel de agarose a 1% contendo brometo de etí dio. A amplificação dos genes foram correlacionadas com a classificação UICC, TNM, graduação histológica, localização e espessura tumoral, invasão de tecidos adjacentes, perineural e vascular. Após seqüenciamento dos “ amplicons" e confirmação dos genes foram confecionadas as sondas de mRNA para realização da técnica de hibridização in situ. Os resultados obtidos através da técnica de RT-PCR mostraram que: a amplificação dos transcritos dos genes HOXB13 e HHEX podem ser detectados tanto no carcinoma epidermóide quanto no tecido não tumoral adjacente à lesão; não existindo diferença na amplificação dos transcritos de ambos genes para os dois grupos de tecido estudados; a amplificação do transcrito do gene HOXB13 mostrou relação estatí stica com os fatores prognósticos: espessura tumoral, invasão perineural e invasão vascular; a amplificação do transcrito do gene HHEX mostrou relação estatí stica com os fatores prognósticos: invasão vascular,envolvimento com os tecidos adjacentes e uma relação inversa com a idade do paciente. A expressão dos transcritos dos genes HOXB13 e HHEX, detectados pela técnica de ISH, mostrou um padrão de marcação consistente e invariável para os tecidos analisados, estando expressos tanto em carcinoma epidermóide quanto no tecido não tumoral adjacentes à lesão. Os resultados apontam para uma correlação entre a expressão do HHEX e do HOXB13 e alguns fatores prognósticos importantes podendo representar um indicador prognóstico valoroso para o entendimento do comportamento biológico do CEB. / The aim of this study was to verify the HOXB13 and HHEX genes expression in oral squamous cell carcinoma (OSCC) using Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) and in situ Hybridization techniques. Tumoral tissues and adjacent non-tumoral oral mucosa specimens were obtained from 30 patients with OSCC at the Head and Neck Surgery Service HC (FMUSP). The samples were cDNA extracted and submitted to the RT-PCR technique. The amplicons were visualized in electrophoresis on a 1% agarose gel with ethidium bromide. Genes expressions were correlated with UICC staging, TNM stage, tumor location, tumor thickness, adjacent tissues involvement, vascular and perineural invasion, and cellular differentiation. Finally, direct sequence analysis was performed on PCR products to confirm cDNA sequence. Riboprobes were confectioned for in situ hybridization analysis. RT-PCR results showed HOXB13 and HHEX transcripts in both tumoral and non-tumoral tissue samples; no statistical correlation was verified between HOXB13/HHEX expressions and tumoral or non-tumoral tissues; there was a positive correlation between HOXB13 tumoral expression and tumor thickness, neural invasion, and vascular invasion; there was a positive correlation between HHEX tumoral expression and adjacent tissue involvement, vascular invasion, and a inverse relationship with patients age; ISH technique exhibited a consistent and invariable pattern of expression for both genes on both tumoral and non-tumoral tissue samples. Present results points out to a correlation between HOXB13 and HHEX expressions, and some important prognostic indicators, and this may represent a valuable tool to understand the biological behavior of OSCC.

Hhex transcription factor is expressed in multiple endoderm-derived tissues, like the liver, where it is essential for proper development. The pleiotropic effect of Hhex in the embryo and its dual role as a transcriptional repressor/activator suggest the presence of different interaction partners capable of modulating its activity and function. In the current study we identified two new Hhex protein interactors: SOX13 and c-Myc. We show that Hhex interacts directly with SOX13. By doing so, Hhex sequesters SOX13 from the SOX13•TCF1 complex, overturning SOX13-dependent repression of the Wnt pathway. On the other hand, Hhex induces proliferation of non-tumorigenic human fibroblast through a Myc-dependent mechanism. Hhex and c-Myc interact directly upregulating Cyclin D1, a c-Myc target gene involved in cell cycle progression and proliferation. Elevation of Cyclin D1 might be the final effector of Hhex capacity to regulate cell proliferation.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19967号 / 医博第4157号 / 新制||医||1017(附属図書館) / 33063 / 京都大学大学院医学研究科医学専攻 / (主査)教授 篠原 隆司, 教授 斎藤 通紀, 教授 山下 潤 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

<p>Most human traits and common diseases have a complex genetic makeup involving more than one gene. The work presented in this thesis investigates standing body height and the common disease type 2 diabetes mellitus (T2DM). In study I we analyzed two single nucleotide polymorphisms (SNPs) in the TCF7L2 gene that had been shown to be associated with T2DM. Analysis was performed in the ULSAM population cohort of ~1500 males. We were able to replicate the association to type 2 diabetes and in addition to that we made a novel find, showing association between the risk alleles and increased proinsulin levels. In study II we analyzed four genes identified to be associated with T2DM in a genome-wide association study. We analyzed SNPs in these genes in the ULSAM population cohort and found an association between SNPs in the HHEX gene and insulin responses and insulin levels. </p><p>The aim of studies III-V was to identify genes affecting normal variation in standing body height. Using a candidate gene approach in study III, 17 genes were screened in the ULSAM population cohort using SNPs. A suggestive association of the ESR1 gene with height was found and confirmed as significant in males from the PIVUS population cohort. In study IV, as a part of the GenomEUtwin project, we performed genetic fine mapping of a linked locus for body height on the X-chromosome. By analyzing 1377 SNPs in 780 Finnish twins, we mapped a region spanning 65kb of this locus with linkage to body height in males. This region contains the GPC3 and PHF6 genes that have known connections to syndromes were standing body height is affected. In study V significant linkage and association to standing body height in males was found for the COL1A11 gene, using population cohorts from Finland and Iceland. </p>

Most human traits and common diseases have a complex genetic makeup involving more than one gene. The work presented in this thesis investigates standing body height and the common disease type 2 diabetes mellitus (T2DM). In study I we analyzed two single nucleotide polymorphisms (SNPs) in the TCF7L2 gene that had been shown to be associated with T2DM. Analysis was performed in the ULSAM population cohort of ~1500 males. We were able to replicate the association to type 2 diabetes and in addition to that we made a novel find, showing association between the risk alleles and increased proinsulin levels. In study II we analyzed four genes identified to be associated with T2DM in a genome-wide association study. We analyzed SNPs in these genes in the ULSAM population cohort and found an association between SNPs in the HHEX gene and insulin responses and insulin levels. The aim of studies III-V was to identify genes affecting normal variation in standing body height. Using a candidate gene approach in study III, 17 genes were screened in the ULSAM population cohort using SNPs. A suggestive association of the ESR1 gene with height was found and confirmed as significant in males from the PIVUS population cohort. In study IV, as a part of the GenomEUtwin project, we performed genetic fine mapping of a linked locus for body height on the X-chromosome. By analyzing 1377 SNPs in 780 Finnish twins, we mapped a region spanning 65kb of this locus with linkage to body height in males. This region contains the GPC3 and PHF6 genes that have known connections to syndromes were standing body height is affected. In study V significant linkage and association to standing body height in males was found for the COL1A11 gene, using population cohorts from Finland and Iceland.