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Functional analysis of a putative HER2-associated expressed enhancer, Her2-Enhancer1, in breast cancer cells

HER-2/neu (HER2) is a member of the epidermal growth factor receptors family, encoding a protein with tyrosine kinase activity. Following the gene amplification or increased HER2 transcription, carcinogenesis has been observed in some cancers. Genetic and epigenetic changes occurring in enhancer seq...

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Autores principales: Rojhannezhad, Mahdieh, Soltani, Bahram M., Vasei, Mohammad, Ghorbanmehr, Nassim, Mowla, Seyed Javad
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10636096/
https://www.ncbi.nlm.nih.gov/pubmed/37945744
http://dx.doi.org/10.1038/s41598-023-46460-x
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author Rojhannezhad, Mahdieh
Soltani, Bahram M.
Vasei, Mohammad
Ghorbanmehr, Nassim
Mowla, Seyed Javad
author_facet Rojhannezhad, Mahdieh
Soltani, Bahram M.
Vasei, Mohammad
Ghorbanmehr, Nassim
Mowla, Seyed Javad
author_sort Rojhannezhad, Mahdieh
collection PubMed
description HER-2/neu (HER2) is a member of the epidermal growth factor receptors family, encoding a protein with tyrosine kinase activity. Following the gene amplification or increased HER2 transcription, carcinogenesis has been observed in some cancers. Genetic and epigenetic changes occurring in enhancer sequences can deeply affect the expression and transcriptional regulation of downstream genes, which can cause some physiological and pathological changes, including tumor progression. A therapeutic approach that directly targets the genomic sequence alterations is of high importance, with low side effects on healthy cells. Here, we employed the CRISPR/Cas9 method to genetically knockout an expressed putative enhancer (GH17J039694; we coined it as Her2-Enhancer1) located within the HER2 gene, 17q12: 39,694,339–39,697,219 (UCSC-hg38). We then investigated the potential regulatory effect of Her2-Enhancer1 on HER2 and HER2-interacting genes. To evaluate the cis and trans effects of Her2-Enhancer1, genetic manipulation of this region was performed in HER2-positive and -negative breast cancer cells. Our bioinformatics and real-time PCR data revealed that this putative enhancer region is indeed expressed, and acts as an expressed enhancer. Further functional analysis on edited and unedited cells revealed a significant alteration in the expression of HER2 variants, as well as some other target genes of HER2. Moreover, the apoptosis rate was considerably elevated within the edited cells. As we expected, Western blot analysis confirmed a reduction in protein levels of HER2, GRB7, the gene interacting with HER2, and P-AKT in the PI3K/AKT pathway. Altogether, our findings revealed an enhancer regulatory role for Her2-Enhancer1 on HER2 and HER2-interacting genes; and that this region has a potential for targeted therapy of HER2-positive cancers.
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spelling pubmed-106360962023-11-11 Functional analysis of a putative HER2-associated expressed enhancer, Her2-Enhancer1, in breast cancer cells Rojhannezhad, Mahdieh Soltani, Bahram M. Vasei, Mohammad Ghorbanmehr, Nassim Mowla, Seyed Javad Sci Rep Article HER-2/neu (HER2) is a member of the epidermal growth factor receptors family, encoding a protein with tyrosine kinase activity. Following the gene amplification or increased HER2 transcription, carcinogenesis has been observed in some cancers. Genetic and epigenetic changes occurring in enhancer sequences can deeply affect the expression and transcriptional regulation of downstream genes, which can cause some physiological and pathological changes, including tumor progression. A therapeutic approach that directly targets the genomic sequence alterations is of high importance, with low side effects on healthy cells. Here, we employed the CRISPR/Cas9 method to genetically knockout an expressed putative enhancer (GH17J039694; we coined it as Her2-Enhancer1) located within the HER2 gene, 17q12: 39,694,339–39,697,219 (UCSC-hg38). We then investigated the potential regulatory effect of Her2-Enhancer1 on HER2 and HER2-interacting genes. To evaluate the cis and trans effects of Her2-Enhancer1, genetic manipulation of this region was performed in HER2-positive and -negative breast cancer cells. Our bioinformatics and real-time PCR data revealed that this putative enhancer region is indeed expressed, and acts as an expressed enhancer. Further functional analysis on edited and unedited cells revealed a significant alteration in the expression of HER2 variants, as well as some other target genes of HER2. Moreover, the apoptosis rate was considerably elevated within the edited cells. As we expected, Western blot analysis confirmed a reduction in protein levels of HER2, GRB7, the gene interacting with HER2, and P-AKT in the PI3K/AKT pathway. Altogether, our findings revealed an enhancer regulatory role for Her2-Enhancer1 on HER2 and HER2-interacting genes; and that this region has a potential for targeted therapy of HER2-positive cancers. Nature Publishing Group UK 2023-11-09 /pmc/articles/PMC10636096/ /pubmed/37945744 http://dx.doi.org/10.1038/s41598-023-46460-x Text en © The Author(s) 2023 https://creativecommons.org/licenses/by/4.0/Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) .
spellingShingle Article
Rojhannezhad, Mahdieh
Soltani, Bahram M.
Vasei, Mohammad
Ghorbanmehr, Nassim
Mowla, Seyed Javad
Functional analysis of a putative HER2-associated expressed enhancer, Her2-Enhancer1, in breast cancer cells
title Functional analysis of a putative HER2-associated expressed enhancer, Her2-Enhancer1, in breast cancer cells
title_full Functional analysis of a putative HER2-associated expressed enhancer, Her2-Enhancer1, in breast cancer cells
title_fullStr Functional analysis of a putative HER2-associated expressed enhancer, Her2-Enhancer1, in breast cancer cells
title_full_unstemmed Functional analysis of a putative HER2-associated expressed enhancer, Her2-Enhancer1, in breast cancer cells
title_short Functional analysis of a putative HER2-associated expressed enhancer, Her2-Enhancer1, in breast cancer cells
title_sort functional analysis of a putative her2-associated expressed enhancer, her2-enhancer1, in breast cancer cells
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10636096/
https://www.ncbi.nlm.nih.gov/pubmed/37945744
http://dx.doi.org/10.1038/s41598-023-46460-x
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