Publicação
Validation of siRNAs against MAP2K1/MEK1 towards colon cancer therapy
| Resumo: | Cancer can be considered a public health problem due to their high incidence and mortality rates. Specifically, colon cancer is the fourth most common and the second deadliest worldwide. The major part of colon cancers occurs due to a family predisposition although some of them can also be related with environmental factors such as smoke, alcohol consumption and a diet rich on fat. Colon cancer therapies depend on the disease stage and rely mostly on surgery and chemotherapy, either adjuvant or neoadjuvant. However, chemotherapy has a few shortcomings as immunosuppression and lack of specificity for instance. Therefore, more specific therapies are need. The use of siRNAs is one of these possibilities since they are able to target specific genes that are often overexpressed on cancer cells. The MAPK pathway plays an important role on cell proliferation, differentiation and apoptosis in mammalian cells. The aberrant activation of this pathway and of MEK1, specifically, is often observed in cancer. So, the goal of this work was the validation of a siRNA targeting MEK1 gene towards colon cancer therapy. MEK1 gene is overexpressed in several colorectal cancer cell lines. Our results showed that the colon cancer cell line with higher expression of MEK1 gene is RKO, so this was the cell line that we used further in the study. RKO cells were transfected with an optimized siRNA concentration of 30 nM and the MEK1 gene knockdown effects on cell cycle and cell proliferation were studied. Moreover, the siRNA transfection efficiency was also evaluated through Western Blot and qRT-PCR. Additionally, it has been reported that MEK1 gene knockdown often leads to a cell cycle arrest on GO-G1 phase. Our results corroborate these reports as a cell cycle arrest on GO-G1 phase was observed, indicating a probable decrease on cell proliferation since the cells do not progress to the S phase. Therefore, SRB and MTS proliferation assays were performed. Although the SRB results were inconclusive, the MTS results confirmed a significant decrease of cell viability on the cells transfected. This could mean that, besides the number of cells remained constant, they may be non-metabolically active. Furthermore, the MEK1_siRNA transfection was considered efficient since it was able to knockdown MEK1 about 52.5% at protein level and about 61.6% at the mRNA level. Additionally, the MEK1 knockdown effect on ERK1 gene, which is located downstream MEK1 gene, was also assessed. Results showed a 52.1% knockdown on the ERK1 gene, thus confirming its effect on cell proliferation. Overall, the results herein gathered are in good agreement with previous reports, thus highlighting the siRNA potential for cancer therapy. |
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| Autores principais: | Carvalho, Ana Luísa Afonso de |
| Assunto: | siRNA MAPK MEK1 Gene silencing Colon cancer Silenciamento de genes Cancro do cólon Engenharia e Tecnologia::Engenharia Médica |
| Ano: | 2018 |
| País: | Portugal |
| Tipo de documento: | dissertação de mestrado |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade do Minho |
| Idioma: | inglês |
| Origem: | RepositóriUM - Universidade do Minho |
| Resumo: | Cancer can be considered a public health problem due to their high incidence and mortality rates. Specifically, colon cancer is the fourth most common and the second deadliest worldwide. The major part of colon cancers occurs due to a family predisposition although some of them can also be related with environmental factors such as smoke, alcohol consumption and a diet rich on fat. Colon cancer therapies depend on the disease stage and rely mostly on surgery and chemotherapy, either adjuvant or neoadjuvant. However, chemotherapy has a few shortcomings as immunosuppression and lack of specificity for instance. Therefore, more specific therapies are need. The use of siRNAs is one of these possibilities since they are able to target specific genes that are often overexpressed on cancer cells. The MAPK pathway plays an important role on cell proliferation, differentiation and apoptosis in mammalian cells. The aberrant activation of this pathway and of MEK1, specifically, is often observed in cancer. So, the goal of this work was the validation of a siRNA targeting MEK1 gene towards colon cancer therapy. MEK1 gene is overexpressed in several colorectal cancer cell lines. Our results showed that the colon cancer cell line with higher expression of MEK1 gene is RKO, so this was the cell line that we used further in the study. RKO cells were transfected with an optimized siRNA concentration of 30 nM and the MEK1 gene knockdown effects on cell cycle and cell proliferation were studied. Moreover, the siRNA transfection efficiency was also evaluated through Western Blot and qRT-PCR. Additionally, it has been reported that MEK1 gene knockdown often leads to a cell cycle arrest on GO-G1 phase. Our results corroborate these reports as a cell cycle arrest on GO-G1 phase was observed, indicating a probable decrease on cell proliferation since the cells do not progress to the S phase. Therefore, SRB and MTS proliferation assays were performed. Although the SRB results were inconclusive, the MTS results confirmed a significant decrease of cell viability on the cells transfected. This could mean that, besides the number of cells remained constant, they may be non-metabolically active. Furthermore, the MEK1_siRNA transfection was considered efficient since it was able to knockdown MEK1 about 52.5% at protein level and about 61.6% at the mRNA level. Additionally, the MEK1 knockdown effect on ERK1 gene, which is located downstream MEK1 gene, was also assessed. Results showed a 52.1% knockdown on the ERK1 gene, thus confirming its effect on cell proliferation. Overall, the results herein gathered are in good agreement with previous reports, thus highlighting the siRNA potential for cancer therapy. |
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