Perfil de expressão de genes associados aos telômeros em carcinoma pulmonar de células não pequenas (NSCLC)
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Data
2018-03-09
Autores
Storti, Camila Baldin
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Universidade Estadual Paulista (Unesp)
Resumo
O câncer de pulmão é o câncer que apresenta maior mortalidade no mundo: >1,6 milhões de óbitos por ano segundo a Organização Mundial de Saúde (OMS), sendo o câncer de pulmão de células não pequenas (NSCLC) o tipo mais frequente (~ 85%) de carcinoma pulmonar. O NSCLC apresenta dois subtipos histológicos principais: adenocarcinoma (LUAD) e carcinoma de células escamosas (LUSC). Pesquisas têm sido realizadas objetivando identificar biomarcadores moleculares úteis como alvo terapêuticos em NSCLC e os telômeros têm sido considerado alvos promissores para terapias antitumoral, devido ao seu papel crucial na integridade, estabilidade e manutenção genômica. Telômeros humanos consistem em DNA repetitivo rico em G associado a proteínas do complexo shelterin e mantido pela ação da telomerase. Estudos recentes mostraram que a expressão de TERT (subunidade catalítica da telomerase) e de alguns componentes do complexo shelterin estão alterados em câncer de pulmão. No entanto, a correlação entre alterações na função telomérica e o desenvolvimento e progressão de NSCLC não foi elucidada. Portanto, o objetivo do presente estudo foi verificar alterações na expressão de genes associados aos telômeros, incluindo os ncRNAs associados à regulação e manutenção dos telômeros em NSCLC, no intuito de identificar novos biomarcadores associados ao desenvolvimento e progressão do NSCLC. Para tanto, foram realizadas análises de expressão de 50 genes associados aos telômeros em amostras de tecido tumoral e tecido pulmonar normal, adjacente ao tumor, de pacientes com NSCLC, utilizando 1) RT-qPCR para análise de expressão em amostras de 28 pacientes, tratados do Hospital das Clinicas da Faculdade de Medicina de Botucatu, 2) Análise de transcritoma utilizando dados de RNA-Seq de amostras de 27 pacientes tratados no Hospital A. C. Camargo e 3) Análise de transcritoma utilizando dados de RNA- Seq, disponíveis no banco de dados The Cancer Genome Atlas (TCGA) e obtidos de amostras de 85 pacientes. Adicionalmente foram analisadas as diferenças entre o perfil de expressão dos genes avaliados em amostras do subtipo LUSC e LUAD. Os resultados revelaram que genes envolvidos com a manutenção dos telômeros estão significativamente, p ≤ 0,05, desregulados nos tumores NSCLC avaliados. Com destaque para os genes TERT, RAD51, DNMT3B, DKC1 e BRCA2 que foram identificados com expressão aumentada nas células tumorais de NSCLC em relação as normais, na maioria das análises. E para os genes SUV39H1, RUVBL1 e DNMT3A que apresentaram aumento de expressão significativo apenas nas amostras do subtipo LUSC. Os ncRNA TERC e TERRA apresentaram expressão elevada nas amostras de NSCLC, destacando a alta expressão de TERC associada ao subtipo LUSC. Somado a estas análises foi calculado o perfil de restrição telomérico (TRF) de amostras tumorais (n=11) e normais adjacentes ao tumor (n=4) de pacientes com NSCLC, e os TRFs de leucócitos de pacientes com NSCLC (n= 14) em comparação com os de doadores saudáveis (n=13). Os telômeros de leucócitos de pacientes com NSCLC estavam menores que os de indivíduos saudáveis (p≤ 0,05) e os telômeros diminuíam conforme a idade do paciente (correlação de Spearman, p= 0,00 e r= -0,644). Porém, não foi encontrada diferença significativa entre os valores de TRF dos tecidos tumorais e normais de pacientes com NSCLC, tal como não foi identificado correlação entre o tamanho de TRF e a idade destes indivíduos, provavelmente devido ao baixo número amostral. Os resultados sugerem que os genes que codificam proteínas e ncRNAs envolvidos com a manutenção dos telômeros podem estar envolvidos na oncogênese de NSCLC e que dentre eles podem haver potenciais biomarcadores para NSCLC, sobretudo do subtipo LUSC, visto a expressão aumentada de alguns genes, SUV39H1, RUVBL1, DNMT3A e TERC, estarem exclusivamente associadas a amostras tumorais deste subtipo.
According to the World Health Organization, lung cancer has a high mortality rate associated with >1.6 million deaths per year, being Non-small cell lung cancer (NSCLC) the most frequent type (~ 85%) of lung carcinoma. Lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) are the two major histological subtypes of NSCLC. Research efforts have been made to identify molecular biomarkers useful as therapeutic targets in NSCLC and telomere have been considered very promising targets for anticancer therapies due to their crucial role in genome integrity maintenance and stability. Human telomeres consist of repetitive G-rich DNA associated with proteins of the shelterin complex, maintained by the action of telomerase. Recent studies showed that expression of TERT (telomerase reverse transcriptase component) and of some of the shelterin components are altered in lung cancer. However, the mechanisms of telomere deregulation associated with NSCLC development and progression have not been elucidated. Therefore, our aim was to study expression changes affecting telomere-associated genes and ncRNAs associated with telomere regulation and maintenance in NSCLC. Such alterations may represent novel biomarkers associated with NSCLC development and progression. For this purpose, expression analyzes of 50 telomere-associated genes were performed in samples of tumor tissue and normal lung tissue, adjacent to the tumor, from patients with NSCLC. The following techniques were used: 1) RT-qPCR for analysis of expression in samples obtained from 28 patients who underwent surgical treatment at Botucatu Clinical Hospital; 2) Transcriptome analysis using RNA-Seq data from samples obtained from 27 patients treated at AC Camargo Hospital 3) Transcriptome analysis using RNA-Seq data, available in the The Cancer Genome Atlas database (TCGA) and obtained from samples of 85 patients. In addition, the differences between the expression profile of the telomere-associated genes in LUSC and LUAD subtype samples were analyzed. The results revealed that many genes involved in telomeres maintenance are significantly, ≤ 0.05, deregulated in the NSCLC tumors. Genes enconding TERT, RAD51, DNMT3B, DKC1 and BRCA2 were overexpressed in NSCLC tumor cells compared to the normal lung tissue. SUV39H1, RUVBL1 and DNMT3A genes were overexpressed only in LUSC subtype samples. The ncRNAs TERC and TERRA also showed increased expression in NSCLC samples, with emphasis to the high expression level of TERC associated with the LUSC subtype In addition, telomere restriction fragment (TRF) was estimated for tumor tissue (n=11) and normal lung tissue (n=4) samples obtained from patients with NSCLC. TRFs from leukocytes obtained from patients with NSCLC (n= 14) were also compared with TRFs obtained from healthy donors (n = 13). The results showed that telomeres of leukocytes obtained from NSCLC patients were shorter than those of healthy donors (p≤0.05) and it decreases according to patient's age (Spearman's correlation, p= 0.00 and r= -0,644). However, no significant difference was found between the TRF obtained from tumor and normal lung tissues of patients with NSCLC, as no correlation was found when comparing TRFs and the age of these individuals, probably due to the low sample size. The results suggest that genes encoding proteins and ncRNAs involved in the maintenance of telomeres may be involved in the oncogenesis of NSCLC, especially the LUSC subtype, since the increased expression of some genes, SUV39H1, RUVBL1, DNMT3A and TERC, are exclusively associated with tumor samples of this subtype.
According to the World Health Organization, lung cancer has a high mortality rate associated with >1.6 million deaths per year, being Non-small cell lung cancer (NSCLC) the most frequent type (~ 85%) of lung carcinoma. Lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) are the two major histological subtypes of NSCLC. Research efforts have been made to identify molecular biomarkers useful as therapeutic targets in NSCLC and telomere have been considered very promising targets for anticancer therapies due to their crucial role in genome integrity maintenance and stability. Human telomeres consist of repetitive G-rich DNA associated with proteins of the shelterin complex, maintained by the action of telomerase. Recent studies showed that expression of TERT (telomerase reverse transcriptase component) and of some of the shelterin components are altered in lung cancer. However, the mechanisms of telomere deregulation associated with NSCLC development and progression have not been elucidated. Therefore, our aim was to study expression changes affecting telomere-associated genes and ncRNAs associated with telomere regulation and maintenance in NSCLC. Such alterations may represent novel biomarkers associated with NSCLC development and progression. For this purpose, expression analyzes of 50 telomere-associated genes were performed in samples of tumor tissue and normal lung tissue, adjacent to the tumor, from patients with NSCLC. The following techniques were used: 1) RT-qPCR for analysis of expression in samples obtained from 28 patients who underwent surgical treatment at Botucatu Clinical Hospital; 2) Transcriptome analysis using RNA-Seq data from samples obtained from 27 patients treated at AC Camargo Hospital 3) Transcriptome analysis using RNA-Seq data, available in the The Cancer Genome Atlas database (TCGA) and obtained from samples of 85 patients. In addition, the differences between the expression profile of the telomere-associated genes in LUSC and LUAD subtype samples were analyzed. The results revealed that many genes involved in telomeres maintenance are significantly, ≤ 0.05, deregulated in the NSCLC tumors. Genes enconding TERT, RAD51, DNMT3B, DKC1 and BRCA2 were overexpressed in NSCLC tumor cells compared to the normal lung tissue. SUV39H1, RUVBL1 and DNMT3A genes were overexpressed only in LUSC subtype samples. The ncRNAs TERC and TERRA also showed increased expression in NSCLC samples, with emphasis to the high expression level of TERC associated with the LUSC subtype In addition, telomere restriction fragment (TRF) was estimated for tumor tissue (n=11) and normal lung tissue (n=4) samples obtained from patients with NSCLC. TRFs from leukocytes obtained from patients with NSCLC (n= 14) were also compared with TRFs obtained from healthy donors (n = 13). The results showed that telomeres of leukocytes obtained from NSCLC patients were shorter than those of healthy donors (p≤0.05) and it decreases according to patient's age (Spearman's correlation, p= 0.00 and r= -0,644). However, no significant difference was found between the TRF obtained from tumor and normal lung tissues of patients with NSCLC, as no correlation was found when comparing TRFs and the age of these individuals, probably due to the low sample size. The results suggest that genes encoding proteins and ncRNAs involved in the maintenance of telomeres may be involved in the oncogenesis of NSCLC, especially the LUSC subtype, since the increased expression of some genes, SUV39H1, RUVBL1, DNMT3A and TERC, are exclusively associated with tumor samples of this subtype.
Descrição
Palavras-chave
Câncer de pulmão de células não pequenas (NSCLC), telômeros, biomarcadores moleculares, genes associados aos telômeros, Non-Small Cell Lung Cancer (NSCLC), telomeres, molecular biomarkers, telomere-associated genes