Variación en los genes codificantes para miRNA-27a y miRNA-423 como variantes de susceptibilidad a cáncer de mama familiar en población chilena
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2017
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es
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Universidad Andrés Bello
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Licencia CC
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El cáncer de mama (CM) es el cáncer más común en mujeres en el mundo. En Chile es la primera causa de mortalidad por cáncer en mujeres (15/100.000 mujeres). Una mejor comprensión de la etiología genética del CM se logró luego del descubrimiento de los genes BRCA1 y BRCA2, sin embargo las mutaciones en estos genes solo dan cuenta del 20-25% de los casos de CM familiar. Actualmente se ha propuesto la existencia de genes de moderada y baja penetrancia que contribuirían a la susceptibilidad del CM familiar en las mujeres BRCA1/2 negativas. Se ha planteado que en los tumores hereditarios, una combinación desfavorable de polimorfismos de nucleótido único (SNPs) presentes en genes de baja penetrancia podría aumentar la susceptibilidad al desarrollo de CM. Evidencia reciente informa que los microRNAs (miRNAs) pueden ser considerados genes de baja penetrancia y que variantes genéticas de tipo SNPs en estos o dentro de su secuencia precursora podrían aumentar la susceptibilidad al CM hereditario. Los miRNAs, son RNAs endógenos no codificantes capaces de regular la expresión génica post-transcripcional al unirse a la región 3’ UTR de un RNAm específico degradando o bloqueando su traducción. En esta tesis se investigó la asociación de los SNPs rs6505162 (Pre-miRNA-423) y rs895819 (pre-miRNA-27a) con el riesgo de CM usando un diseño caso-control en 807 controles y 440 casos de CM BRCA1/2 negativo familiar o diagnóstico temprano de la población Chilena. El rs6505162 C>A se asoció significativamente con un mayor riesgo de CM familiar en pacientes con una fuerte historia familiar de CM (OR=1,7 [IC del 95% 1,0-2,0] p=0,05). Para el rs895819 A>G, el genotipo G /G se asoció significativamente con una reducción del riesgo para CM en familias con un historial moderado de CM (OR=0,3 [IC del 95%: 0,1-0,8] p=0,01). Posteriormente, cuantificamos los niveles de expresión de las hebras 3p y 5p de los miRNA-423 y miRNA-27a a través de TaqMan Real-Time PCR en tres líneas celulares de mama, MCF-7, MDA-MB-231 y MCF-10F, en presencia del alelo normal o del alelo polimórfico. La línea celular MCF-10F mostró una sobreexpresión de la hebra 5p tanto del miRNA-423 como del miRNA-27a cuando se transfectó con los vectores que portaban el alelo normal en la secuencia del pre-miRNA. Se detectó una sobreexpresión de la hebra 3p y 5p en las líneas celulares MCF-7 y MDA-MB-231 cuando las células fueron transfectadas con el vector pre-miRNA-423-A (alelo polimórfico). Los niveles de expresión de las hebras 3p y 5p fueron significativamente mayores en la línea celular MCF-7 cuando las células fueron transfectadas con el vector de expresión pre-miRNA-27a-G (alelo polimórfico). Los resultados para la línea celular MDA-MB-231 no fueron significativos.
Breast cancer (BC) is the most common cancer in women in the world. In Chile it is the leading cause of cancer mortality in women (15/100,000 women). A better understanding of the genetic etiology of BC was achieved after the discovery of BRCA1 and BRCA2 genes, however mutations in these genes account for only 20-25% of familial BC cases. It has been proposed the existence of genes of moderate and low penetrance that would contribute to the susceptibility of familial BC in BRCA1/2 negative women. It has been suggested that in hereditary tumors, an unfavorable combination of single nucleotide polymorphisms (SNPs) present in low penetrance genes could increase susceptibility to BC development. Recent evidence reports that microRNAs (miRNAs) may be considered low penetrance genes and those genetic variants of SNPs in these or within their precursor sequence could increase susceptibility to hereditary BC. MiRNAs are endogenous non-coding RNAs capable of regulating post-transcriptional gene expression by binding to the 3 'UTR region of a specific mRNA by degrading or blocking its translation. In this thesis we investigated the association of SNPs rs6505162 (Pre-miRNA-423) and rs895819 (pre-miRNA-27a) with BC risk using a case-control design in 807 controls and 440 cases of BC BRCA1/2 negative family or early diagnosis of the Chilean population. The rs6505162 C>A was significantly associated with an increased risk of familial BC in patients with a strong family history of BC (OR=1.7 [95% CI 1.3-.2.0] p=0.05). For the SNP rs895819, G/G genotype was significantly associated with a reduced risk for BC in families with a moderate history of BC (OR=0.3 [95% CI 0.1-0.8] p=0.01). We then quantified the expression levels of the 3p and 5p strands of miRNA-423 and miRNA-27a through TaqMan Real-Time PCR in three breast cell lines, MCF-7, MDA- MB-231 and MCF-10F in the presence of the normal allele or the polymorphic allele The MCF-10F cell line showed overexpression of the 5p strand of both miRNA-423 and miRNA-27a when transfected with the vectors carrying the allele in the pre-miRNA sequence Overexpression of the 3p and 5p strand was detected in MCF-7 and MDA-MB-231 cell lines when cells were transfected with the pre-miRNA-423-A vector (allele polymorphic.) The expression levels of strands 3p and 5p were significantly higher in the MCF-7 cell line when cells were transfected with n the pre-miRNA-27a-G expression vector (polymorphic allele). The results for MDA-MB-231 were not significant.
Breast cancer (BC) is the most common cancer in women in the world. In Chile it is the leading cause of cancer mortality in women (15/100,000 women). A better understanding of the genetic etiology of BC was achieved after the discovery of BRCA1 and BRCA2 genes, however mutations in these genes account for only 20-25% of familial BC cases. It has been proposed the existence of genes of moderate and low penetrance that would contribute to the susceptibility of familial BC in BRCA1/2 negative women. It has been suggested that in hereditary tumors, an unfavorable combination of single nucleotide polymorphisms (SNPs) present in low penetrance genes could increase susceptibility to BC development. Recent evidence reports that microRNAs (miRNAs) may be considered low penetrance genes and those genetic variants of SNPs in these or within their precursor sequence could increase susceptibility to hereditary BC. MiRNAs are endogenous non-coding RNAs capable of regulating post-transcriptional gene expression by binding to the 3 'UTR region of a specific mRNA by degrading or blocking its translation. In this thesis we investigated the association of SNPs rs6505162 (Pre-miRNA-423) and rs895819 (pre-miRNA-27a) with BC risk using a case-control design in 807 controls and 440 cases of BC BRCA1/2 negative family or early diagnosis of the Chilean population. The rs6505162 C>A was significantly associated with an increased risk of familial BC in patients with a strong family history of BC (OR=1.7 [95% CI 1.3-.2.0] p=0.05). For the SNP rs895819, G/G genotype was significantly associated with a reduced risk for BC in families with a moderate history of BC (OR=0.3 [95% CI 0.1-0.8] p=0.01). We then quantified the expression levels of the 3p and 5p strands of miRNA-423 and miRNA-27a through TaqMan Real-Time PCR in three breast cell lines, MCF-7, MDA- MB-231 and MCF-10F in the presence of the normal allele or the polymorphic allele The MCF-10F cell line showed overexpression of the 5p strand of both miRNA-423 and miRNA-27a when transfected with the vectors carrying the allele in the pre-miRNA sequence Overexpression of the 3p and 5p strand was detected in MCF-7 and MDA-MB-231 cell lines when cells were transfected with the pre-miRNA-423-A vector (allele polymorphic.) The expression levels of strands 3p and 5p were significantly higher in the MCF-7 cell line when cells were transfected with n the pre-miRNA-27a-G expression vector (polymorphic allele). The results for MDA-MB-231 were not significant.
Notas
Tesis (Magíster en Biotecnología)
Palabras clave
Genes, Cáncer Mamario