Quantificação do RNA mensageiro de tiroglobulina, do receptor do hormônio tiroestimulante e de calcitonina obtidos do sangue periférico de uma população com função tiroidiana normal
Data
2014-03-31
Autores
Melo, Maria Clara de Carvalho 
Orientadores
Maciel, Rui Monteiro de Barros
Tipo
Dissertação de mestrado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
O câncer de tiroide é a neoplasia endócrina mais comum. No seguimento do carcinoma diferenciado de tiroide (CDT), alguns grupos têm buscado novas ferramentas moleculares, por exemplo, a utilização do mRNA-TG e TSHR como alternativa as dificuldades de dosagem sérica de tiroglobulina (sTg) na presença de anticorpo anti-Tg circulante em até 25% dos pacientes. No caso do carcinoma medular de tiroide (CMT), estudos apontam que alterações na expressão do gene da calcitonina (CALCA) antecedem o aparecimento de tumores em ratos, sugerindo que elevações na quantificação do mRNA-CALCA no ser humano poderia ser um marcador mais precoce de doença.Neste trabalho quantificamos a expressão do mRNA-TG, TSHR e CALCA circulante e correlacionamos essa medida com a dosagem de sérica de Tg, TSH e calcitonina (sCT) numa população controle sem evidência de doença tiroidiana com intuito de estabelecer valor de referência normal basal. Estudamos 66 voluntários provenientes da Universidade Federal de São Paulo, sendo 38 mulheres e 28 homens. TSH, sTg, anti-Tg e sCT foram dosados por métodos imunofluorométricos previamente desenvolvidos em nosso laboratório. T4L e anti-TPO foram dosados por eletroquimioluminescência utilizando kits comercias. Avaliamos a extração de RNA de sangue periférico por 4 métodos. A quantificação foi realizada através da curva padrão absoluta (cDNA plasmidial) para mRNA-TG e TSHR e de forma relativa para CALCA. Entre os métodos, Trizol LS apresentou melhor rendimento de RNA total. Voluntários da população controle apresentaram média de TSH de 1,7 ± 0,9UI/mL (0,4 - 4,41), T4 livre de 1,23 ± 0,14 ng/dL (0,87 - 1,51) e de sTg de 14,62 ±11,5 ng/mL (0,5 – 61,7). Anti-Tg e anti-TPO foram todos negativos. A média do mRNA-TG foi de 0,45 ± 0,20 pg mRNA-TG/µL (0,193 a 1,023) e TSHR de 0,079 ± 0,01 µg mRNA-TSHR/µL (0,0381 a 0,123). Doze voluntários apresentaram mRNA CALCA indetectável e a média da sCt destes voluntários foi de 4,95 ± 3,79 pg/mL (1 a 12,8). Dos 7 detectáveis, os valores do mRNA-CALCA variaram de zero a 1,23 e sCT de 1 a 10,4 ± 4,91 pg/mL. Não observamos correlação direta entre a medida sérica das proteínas basais (sTg, TSH e sCT) e seus respectivos transcritos.Tendo em vista que quantificamos mRNA-TG e TSHR por RT-qPCR em sangue periférico de uma população controle, esta metodologia não deve ser utilizada como biomarcador específico no diagnóstico e seguimento de câncer de tiroide. Portanto, antevemos a necessidade de mais estudos de desenvolvimento de curvas de quantificação
de mRNA circulante com objetivo de obter valores de referência para grupos de indivíduos sem e com doença tiroidiana quer seja autoimune ou neoplásica. Apesar de não ter sido mostrado correlação entre mRNA-CALCA e sCT, a quantificação de mRNA-CALCA circulante em sangue periférico pode refinar o seguimento de pacientes com CMT.
Thyroid cancer is the most common endocrine neoplasia. A few groups have investigated new molecular tools for the follow-up of differentiated thyroid carcinoma (DTC), for example, the use of TG and TSHR-mRNA as an alternative methodology due to the troublesome measurement of seric thyroglobulin (sTg) given the prevalence of circulatory anti-Tg antibody in up to 25% of all patients analysed. For medullary thyroid carcinoma (MTC), the literature outlines that alterations in the calcitonin gene (CALCA) precede tumour outbreak in rats, suggesting that increased CALCA-mRNA measurement in humans could become an earlier disease biomarker. At this study, we quantified the expression of circulating TG, TSHR and CALCA mRNAs and correlated such measurement with the seric quantification of Tg, TSH and calcitonin (sCT) at our control population with no evidence of thyroid disease. Our aim was to establish a ‘cut-off’ value as reference for normal basal levels. We investigated such markers at 66 volunteers from Universidade Federal de São Paulo, being 38 women and 28 men. TSH, sTg, anti-Tg and sCT were measured via ‘in house’ imunofluorimetric assays previously developed at our laboratory. Free T4 and anti-TPO were quantified by commercial eletrochemiluminescence kits. We evaluated peripheral blood RNA extraction via 4 methods. Quantification was performed via absolute standard curve (plasmidial cDNA) for TG and TSHR-mRNAs, and relative qPCR for CALCA-mRNA. From such aforementioned methods, Trizol LS presented best total RNA output. Volunteers from control population presented TSH mean of 1.7 ± 0.9 UI/mL (0.4 – 4.41), free T4 of 1.23 ± 0.14 ng/dL (0.87 – 1.51) and sTg of 14.62 ±11.5 ng/mL (0.5 – 61.7). Anti-Tg and anti-TPO were all negative. The mean for TG-mRNA was 0.45 ± 0.20 pg mRNA-TG/µL (0.193 a 1.023) and for TSHR was 0.079 ± 0.01 µg mRNA-TSHR/µL (0.0381 a 0.123). Twelve volunteers presented undetectable mRNA-CALCA and their mean mRNA sCT was 4.95 ± 3.79 pg/mL (1 a 12.8). Seven detectable volunteers’ mRNA-CALCA values varied from zero to 1.23 and sCT from 1 to 10.4 ± 4,91 pg/mL. We did not observe direct correlation between seric measurement of basal proteins (sTg, TSH and sCt) and their respective transcripts. Given that we quantified TG and TSHR mRNAs via RT-qPCR in peripheral blood samples from our contorl population, this methodology should be used for such specific biomarkers for thyroid cancer diagnosis and follow-up. Therefore, we foresee the need for more studies to develop quantification curves of circulating mRNA in order to obtain reference values for those people affected and free of thyroid disease, be it autoimune or neoplastic. Finally, although there was no correlation between CALCA and sCT mRNAs, the circulating CALCA-mRNA measurement in peripheral blood samples may refine MTC patients’ follow-up.
Thyroid cancer is the most common endocrine neoplasia. A few groups have investigated new molecular tools for the follow-up of differentiated thyroid carcinoma (DTC), for example, the use of TG and TSHR-mRNA as an alternative methodology due to the troublesome measurement of seric thyroglobulin (sTg) given the prevalence of circulatory anti-Tg antibody in up to 25% of all patients analysed. For medullary thyroid carcinoma (MTC), the literature outlines that alterations in the calcitonin gene (CALCA) precede tumour outbreak in rats, suggesting that increased CALCA-mRNA measurement in humans could become an earlier disease biomarker. At this study, we quantified the expression of circulating TG, TSHR and CALCA mRNAs and correlated such measurement with the seric quantification of Tg, TSH and calcitonin (sCT) at our control population with no evidence of thyroid disease. Our aim was to establish a ‘cut-off’ value as reference for normal basal levels. We investigated such markers at 66 volunteers from Universidade Federal de São Paulo, being 38 women and 28 men. TSH, sTg, anti-Tg and sCT were measured via ‘in house’ imunofluorimetric assays previously developed at our laboratory. Free T4 and anti-TPO were quantified by commercial eletrochemiluminescence kits. We evaluated peripheral blood RNA extraction via 4 methods. Quantification was performed via absolute standard curve (plasmidial cDNA) for TG and TSHR-mRNAs, and relative qPCR for CALCA-mRNA. From such aforementioned methods, Trizol LS presented best total RNA output. Volunteers from control population presented TSH mean of 1.7 ± 0.9 UI/mL (0.4 – 4.41), free T4 of 1.23 ± 0.14 ng/dL (0.87 – 1.51) and sTg of 14.62 ±11.5 ng/mL (0.5 – 61.7). Anti-Tg and anti-TPO were all negative. The mean for TG-mRNA was 0.45 ± 0.20 pg mRNA-TG/µL (0.193 a 1.023) and for TSHR was 0.079 ± 0.01 µg mRNA-TSHR/µL (0.0381 a 0.123). Twelve volunteers presented undetectable mRNA-CALCA and their mean mRNA sCT was 4.95 ± 3.79 pg/mL (1 a 12.8). Seven detectable volunteers’ mRNA-CALCA values varied from zero to 1.23 and sCT from 1 to 10.4 ± 4,91 pg/mL. We did not observe direct correlation between seric measurement of basal proteins (sTg, TSH and sCt) and their respective transcripts. Given that we quantified TG and TSHR mRNAs via RT-qPCR in peripheral blood samples from our contorl population, this methodology should be used for such specific biomarkers for thyroid cancer diagnosis and follow-up. Therefore, we foresee the need for more studies to develop quantification curves of circulating mRNA in order to obtain reference values for those people affected and free of thyroid disease, be it autoimune or neoplastic. Finally, although there was no correlation between CALCA and sCT mRNAs, the circulating CALCA-mRNA measurement in peripheral blood samples may refine MTC patients’ follow-up.
Descrição
Citação
MELO, Maria Clara de Carvalho. Quantificação do RNA mensageiro de tiroglobulina, do receptor do hormônio tiroestimulante e de calcitonina obtidos do sangue periférico de uma população com função tiroidiana normal. São Paulo, 2014. 107 f. Dissertação (Mestrado em Medicina Translacional) – Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, 2014.