Study of Inflammation in Colorectal Cancer
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2019-01-29
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Globalmente, tanto em países economicamente desenvolvidos como em desenvolvimento, o cancro tornou-se um dos maiores problemas da sociedade hoje em dia. O Cancro colorretal é dos mais incidentes e frequentes na Europa. A proteína inibitória de macrófagos (MIF) é uma citocina envolvida na imunidade célular, imunorregulação e inflamação. É capaz de desencadear respostas imunes significativas autócrina e/ou parácrinamente através da indução de citocinas próinflamatórias. Como mediador pró-inflamatório, o MIF tem sido demonstrado como estando envolvido no cancro. O gene MIF possui um SNP putativamente funcional, transversão de G>C na região flanqueadora do gene (rs755622), que tem sido reportado como associado ao cancro colorretal (CCR) e outras doenças. Primeiramente, extraímos DNA de 172 amostras de sangue usando buffy coat. Posteriormente, procedeu-se à genotipagem das amostras extraídas utilizando-se a técnica de PCR em tempo real, com recurso a sondas Taqman, otimizadas para este SNP. Seguiu-se depois para sequenciação utilizando a técnica de Sanger para validação dos resultados. Obtivemos as seguintes frequências: GG 70%; GC 28%; CC 2%; consistente com frequências alélicas de 84% G e 16% C (similares às frequências alélicas presente na base de dados Ensembl: 81% -G; 19% -C). Após análise estatística entre o polimorfismo do MIF e os dados clínico-patológicos, não foi encontrada correlação significativa. Relativamente à técnica de imunohistoquímica também foi realizada em 32 amostras, não apenas para caracterizar a proteína MIF que foi o ponto focal, mas também na tentativa de estabelecer uma comparação entre MIF, macrófagos e a infiltração linfocitária em tumores utilizamos um anticorpo para a proteína MIF, outro para CD68 (receptor de macrófagos) e outro para CD3 (co-receptor de células T). Análises de genótipo-fenótipo foram realizadas entre TAMs, TILs e os genótipos MIF -173 G> C onde nenhuma significância foi encontrada. Em relação à intensidade e percentagem de células coradas com MIF, foram então correlacionados com os genótipos MIF -173 G> C,onde se encontrou significância na variável modelo aditivo quando referente à intensidade de coloração. O principal objetivo foi correlacionar todos os dados clinicopatológicos, genéticos e imunohistoquímicos, a fim de obter dados / conclusões que possam vir a ser úteis para a prática clínica e melhorar o tratamento e a sobrevida do paciente no futuro
Worldwide, in both economically developed and developing countries, cancer has become one of the biggest problems in society and colorectal cancer is the second most incident and most frequent cause of death in Europe. MIF (macrophage inhibitory factor) protein is a cytokine involved in cell-mediated immunity, immunoregulation and inflammation. It is capable of triggering significant immune responses through autocrine/paracrine loops via the induction of pro-inflammatory cytokines. As a pro-inflammatory mediator, MIF has been implicated in cancer. MIF gene has a putatively functional SNP, G-to-C transversion at the 5’-flanking region (rs755622), which has been reported to be associated with Colorectal Cancer (CRC) and other diseases. Firstly, DNA was extracted from 172 blood samples using its buffy coat. Thereafter we proceeded to the genotyping of the extracted samples using real time PCR technique using Taqman probes optimized for this SNP, followed by Sanger sequencing for result confirmation. We obtained the following frequencies: GG 70%; GC 28%; CC 2%; consistent with an 84% G and 16% C allelic frequencies (similar to Ensembl database allelic frequencies: 81%-G; 19%-C). After statistical analysis between clinicopathological parameters and MIF polymorphism, no significant correlation was found. Immunohistochemistry technique using an antibody for MIF, CD68 (macrophage receptor) and for CD3 (T cell co-receptor) was also optimized in 32 case samples, not only to characterize the MIF protein, which was the focal point, but also in an attempt to establish a comparison between the MIF and the macrophage and lymphocyte infiltration in tumors. Genotype-phenotype analyses was performed between TAMs, TILs and MIF -173 G>C genotypes using Mann-Whitney U tests, and no significance was found. Concerning intensity and percentage of cells stained with MIF were then correlated with MIF -173 G>C genotypes using Fisher’s exact and Pearson chi-square test. It was found significance in the additive and recessive model variables. We aimed to establish correlations between all clinicopathological, genetic and immunohistochemistry data in order to draw data/conclusions that could possibly be useful for clinical practice and improve patient treatment and survival in the future.
Worldwide, in both economically developed and developing countries, cancer has become one of the biggest problems in society and colorectal cancer is the second most incident and most frequent cause of death in Europe. MIF (macrophage inhibitory factor) protein is a cytokine involved in cell-mediated immunity, immunoregulation and inflammation. It is capable of triggering significant immune responses through autocrine/paracrine loops via the induction of pro-inflammatory cytokines. As a pro-inflammatory mediator, MIF has been implicated in cancer. MIF gene has a putatively functional SNP, G-to-C transversion at the 5’-flanking region (rs755622), which has been reported to be associated with Colorectal Cancer (CRC) and other diseases. Firstly, DNA was extracted from 172 blood samples using its buffy coat. Thereafter we proceeded to the genotyping of the extracted samples using real time PCR technique using Taqman probes optimized for this SNP, followed by Sanger sequencing for result confirmation. We obtained the following frequencies: GG 70%; GC 28%; CC 2%; consistent with an 84% G and 16% C allelic frequencies (similar to Ensembl database allelic frequencies: 81%-G; 19%-C). After statistical analysis between clinicopathological parameters and MIF polymorphism, no significant correlation was found. Immunohistochemistry technique using an antibody for MIF, CD68 (macrophage receptor) and for CD3 (T cell co-receptor) was also optimized in 32 case samples, not only to characterize the MIF protein, which was the focal point, but also in an attempt to establish a comparison between the MIF and the macrophage and lymphocyte infiltration in tumors. Genotype-phenotype analyses was performed between TAMs, TILs and MIF -173 G>C genotypes using Mann-Whitney U tests, and no significance was found. Concerning intensity and percentage of cells stained with MIF were then correlated with MIF -173 G>C genotypes using Fisher’s exact and Pearson chi-square test. It was found significance in the additive and recessive model variables. We aimed to establish correlations between all clinicopathological, genetic and immunohistochemistry data in order to draw data/conclusions that could possibly be useful for clinical practice and improve patient treatment and survival in the future.
Descrição
Master’s Dissertation in Biotechnology for Health Sciences
Palavras-chave
MIF , colorretal , genotipagem , Imunohistoquímica , SNP