Identification of AGR2 Gene-Specific Expression Patterns Associated with Epithelial-Mesenchymal Transition
The TGF-β signaling pathway is involved in numerous cellular processes, and its deregulation may result in cancer development. One of the key processes in tumor progression and metastasis is epithelial to mesenchymal transition (EMT), in which TGF-β signaling plays important roles. Recently, AGR2 wa...
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MDPI AG
2022-09-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/23/18/10845 |
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| author | Andrea Martisova Lucia Sommerova Adam Krejci Iveta Selingerova Tamara Kolarova Filip Zavadil Kokas Milos Holanek Jan Podhorec Tomas Kazda Roman Hrstka |
| author_facet | Andrea Martisova Lucia Sommerova Adam Krejci Iveta Selingerova Tamara Kolarova Filip Zavadil Kokas Milos Holanek Jan Podhorec Tomas Kazda Roman Hrstka |
| author_sort | Andrea Martisova |
| collection | DOAJ |
| description | The TGF-β signaling pathway is involved in numerous cellular processes, and its deregulation may result in cancer development. One of the key processes in tumor progression and metastasis is epithelial to mesenchymal transition (EMT), in which TGF-β signaling plays important roles. Recently, AGR2 was identified as a crucial component of the cellular machinery responsible for maintaining the epithelial phenotype, thereby interfering with the induction of mesenchymal phenotype cells by TGF-β effects in cancer. Here, we performed transcriptomic profiling of A549 lung cancer cells with CRISPR-Cas9 mediated <i>AGR2</i> knockout with and without TGF-β treatment. We identified significant changes in transcripts associated with focal adhesion and eicosanoid production, in particular arachidonic acid metabolism. Changes in transcripts associated with the focal adhesion pathway were validated by RT-qPCR of <i>COL4A1</i>, <i>COL4A2</i>, <i>FLNA</i>, <i>VAV3</i>, <i>VEGFA</i>, and <i>VINC</i> mRNAs. In addition, immunofluorescence showed the formation of stress fibers and vinculin foci in cells without AGR2 and in response to TGF-β treatment, with synergistic effects observed. These findings imply that both AGR2 downregulation and TGF-β have a role in focal adhesion formation and cancer cell migration and invasion. Transcripts associated with arachidonic acid metabolism were downregulated after both <i>AGR2</i> knockout and TGF-β treatment and were validated by RT-qPCR of <i>GPX2</i>, <i>PTGS2</i>, and <i>PLA2G4A</i>. Since PGE<sub>2</sub> is a product of arachidonic acid metabolism, its lowered concentration in media from <i>AGR2</i>-knockout cells was confirmed by ELISA. Together, our results demonstrate that AGR2 downregulation and TGF-β have an essential role in focal adhesion formation; moreover, we have identified AGR2 as an important component of the arachidonic acid metabolic pathway. |
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| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-09T23:42:41Z |
| publishDate | 2022-09-01 |
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| spelling | doaj.art-3180081033df4b7b973492ac34e9ebd22023-11-23T16:49:05ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672022-09-0123181084510.3390/ijms231810845Identification of AGR2 Gene-Specific Expression Patterns Associated with Epithelial-Mesenchymal TransitionAndrea Martisova0Lucia Sommerova1Adam Krejci2Iveta Selingerova3Tamara Kolarova4Filip Zavadil Kokas5Milos Holanek6Jan Podhorec7Tomas Kazda8Roman Hrstka9Research Centre for Applied Molecular Oncology (RECAMO), Masaryk Memorial Cancer Institute, Zluty Kopec 7, 65653 Brno, Czech RepublicResearch Centre for Applied Molecular Oncology (RECAMO), Masaryk Memorial Cancer Institute, Zluty Kopec 7, 65653 Brno, Czech RepublicResearch Centre for Applied Molecular Oncology (RECAMO), Masaryk Memorial Cancer Institute, Zluty Kopec 7, 65653 Brno, Czech RepublicResearch Centre for Applied Molecular Oncology (RECAMO), Masaryk Memorial Cancer Institute, Zluty Kopec 7, 65653 Brno, Czech RepublicResearch Centre for Applied Molecular Oncology (RECAMO), Masaryk Memorial Cancer Institute, Zluty Kopec 7, 65653 Brno, Czech RepublicResearch Centre for Applied Molecular Oncology (RECAMO), Masaryk Memorial Cancer Institute, Zluty Kopec 7, 65653 Brno, Czech RepublicDepartment of Comprehensive Cancer Care, Masaryk Memorial Cancer Institute, Zluty Kopec 7, 65653 Brno, Czech RepublicDepartment of Comprehensive Cancer Care, Masaryk Memorial Cancer Institute, Zluty Kopec 7, 65653 Brno, Czech RepublicResearch Centre for Applied Molecular Oncology (RECAMO), Masaryk Memorial Cancer Institute, Zluty Kopec 7, 65653 Brno, Czech RepublicResearch Centre for Applied Molecular Oncology (RECAMO), Masaryk Memorial Cancer Institute, Zluty Kopec 7, 65653 Brno, Czech RepublicThe TGF-β signaling pathway is involved in numerous cellular processes, and its deregulation may result in cancer development. One of the key processes in tumor progression and metastasis is epithelial to mesenchymal transition (EMT), in which TGF-β signaling plays important roles. Recently, AGR2 was identified as a crucial component of the cellular machinery responsible for maintaining the epithelial phenotype, thereby interfering with the induction of mesenchymal phenotype cells by TGF-β effects in cancer. Here, we performed transcriptomic profiling of A549 lung cancer cells with CRISPR-Cas9 mediated <i>AGR2</i> knockout with and without TGF-β treatment. We identified significant changes in transcripts associated with focal adhesion and eicosanoid production, in particular arachidonic acid metabolism. Changes in transcripts associated with the focal adhesion pathway were validated by RT-qPCR of <i>COL4A1</i>, <i>COL4A2</i>, <i>FLNA</i>, <i>VAV3</i>, <i>VEGFA</i>, and <i>VINC</i> mRNAs. In addition, immunofluorescence showed the formation of stress fibers and vinculin foci in cells without AGR2 and in response to TGF-β treatment, with synergistic effects observed. These findings imply that both AGR2 downregulation and TGF-β have a role in focal adhesion formation and cancer cell migration and invasion. Transcripts associated with arachidonic acid metabolism were downregulated after both <i>AGR2</i> knockout and TGF-β treatment and were validated by RT-qPCR of <i>GPX2</i>, <i>PTGS2</i>, and <i>PLA2G4A</i>. Since PGE<sub>2</sub> is a product of arachidonic acid metabolism, its lowered concentration in media from <i>AGR2</i>-knockout cells was confirmed by ELISA. Together, our results demonstrate that AGR2 downregulation and TGF-β have an essential role in focal adhesion formation; moreover, we have identified AGR2 as an important component of the arachidonic acid metabolic pathway.https://www.mdpi.com/1422-0067/23/18/10845AGR2EMTTGF-βRNAseqarachidonic acidfocal adhesion |
| spellingShingle | Andrea Martisova Lucia Sommerova Adam Krejci Iveta Selingerova Tamara Kolarova Filip Zavadil Kokas Milos Holanek Jan Podhorec Tomas Kazda Roman Hrstka Identification of AGR2 Gene-Specific Expression Patterns Associated with Epithelial-Mesenchymal Transition International Journal of Molecular Sciences AGR2 EMT TGF-β RNAseq arachidonic acid focal adhesion |
| title | Identification of AGR2 Gene-Specific Expression Patterns Associated with Epithelial-Mesenchymal Transition |
| title_full | Identification of AGR2 Gene-Specific Expression Patterns Associated with Epithelial-Mesenchymal Transition |
| title_fullStr | Identification of AGR2 Gene-Specific Expression Patterns Associated with Epithelial-Mesenchymal Transition |
| title_full_unstemmed | Identification of AGR2 Gene-Specific Expression Patterns Associated with Epithelial-Mesenchymal Transition |
| title_short | Identification of AGR2 Gene-Specific Expression Patterns Associated with Epithelial-Mesenchymal Transition |
| title_sort | identification of agr2 gene specific expression patterns associated with epithelial mesenchymal transition |
| topic | AGR2 EMT TGF-β RNAseq arachidonic acid focal adhesion |
| url | https://www.mdpi.com/1422-0067/23/18/10845 |
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