Evaluation of a Novel Plasmid for Simultaneous Gene Electrotransfer-Mediated Silencing of CD105 and CD146 in Combination with Irradiation
Targeting tumor vasculature through specific endothelial cell markers represents a promising approach for cancer treatment. Here our aim was to construct an antibiotic resistance gene-free plasmid encoding shRNAs to simultaneously target two endothelial cell markers, CD105 and CD146, and to test its...
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| Format: | Article |
| Language: | English |
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MDPI AG
2021-03-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/22/6/3069 |
| _version_ | 1797541029991153664 |
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| author | Monika Savarin Urska Kamensek Katarina Znidar Vesna Todorovic Gregor Sersa Maja Cemazar |
| author_facet | Monika Savarin Urska Kamensek Katarina Znidar Vesna Todorovic Gregor Sersa Maja Cemazar |
| author_sort | Monika Savarin |
| collection | DOAJ |
| description | Targeting tumor vasculature through specific endothelial cell markers represents a promising approach for cancer treatment. Here our aim was to construct an antibiotic resistance gene-free plasmid encoding shRNAs to simultaneously target two endothelial cell markers, CD105 and CD146, and to test its functionality and therapeutic potential in vitro when delivered by gene electrotransfer (GET) and combined with irradiation (IR). Functionality of the plasmid was evaluated by determining the silencing of the targeted genes using qRT-PCR. Antiproliferative and antiangiogenic effects were determined by the cytotoxicity assay tube formation assay and wound healing assay in murine endothelial cells 2H-11. The functionality of the plasmid construct was also evaluated in malignant melanoma tumor cell line B16F10. Additionally, potential activation of immune response was measured by induction of DNA sensor STING and proinflammatory cytokines by qRT-PCR in endothelial cells 2H-11. We demonstrated that the plasmid construction was successful and can efficiently silence the expression of the two targeted genes. As a consequence of silencing, reduced migration rate and angiogenic potential was confirmed in 2H-11 endothelial cells. Furthermore, induction of DNA sensor STING and proinflammatory cytokines were determined, which could add to the therapeutic effectiveness when used in vivo. To conclude, we successfully constructed a novel plasmid DNA with two shRNAs, which holds a great promise for further in vivo testing. |
| first_indexed | 2024-03-10T13:09:29Z |
| format | Article |
| id | doaj.art-31807bb43787496e95f1cdecc3b71eb7 |
| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-10T13:09:29Z |
| publishDate | 2021-03-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | International Journal of Molecular Sciences |
| spelling | doaj.art-31807bb43787496e95f1cdecc3b71eb72023-11-21T10:51:03ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672021-03-01226306910.3390/ijms22063069Evaluation of a Novel Plasmid for Simultaneous Gene Electrotransfer-Mediated Silencing of CD105 and CD146 in Combination with IrradiationMonika Savarin0Urska Kamensek1Katarina Znidar2Vesna Todorovic3Gregor Sersa4Maja Cemazar5Department of Experimental Oncology, Institute of Oncology Ljubljana, 1000 Ljubljana, SloveniaDepartment of Experimental Oncology, Institute of Oncology Ljubljana, 1000 Ljubljana, SloveniaDepartment of Experimental Oncology, Institute of Oncology Ljubljana, 1000 Ljubljana, SloveniaDepartment of Experimental Oncology, Institute of Oncology Ljubljana, 1000 Ljubljana, SloveniaDepartment of Experimental Oncology, Institute of Oncology Ljubljana, 1000 Ljubljana, SloveniaDepartment of Experimental Oncology, Institute of Oncology Ljubljana, 1000 Ljubljana, SloveniaTargeting tumor vasculature through specific endothelial cell markers represents a promising approach for cancer treatment. Here our aim was to construct an antibiotic resistance gene-free plasmid encoding shRNAs to simultaneously target two endothelial cell markers, CD105 and CD146, and to test its functionality and therapeutic potential in vitro when delivered by gene electrotransfer (GET) and combined with irradiation (IR). Functionality of the plasmid was evaluated by determining the silencing of the targeted genes using qRT-PCR. Antiproliferative and antiangiogenic effects were determined by the cytotoxicity assay tube formation assay and wound healing assay in murine endothelial cells 2H-11. The functionality of the plasmid construct was also evaluated in malignant melanoma tumor cell line B16F10. Additionally, potential activation of immune response was measured by induction of DNA sensor STING and proinflammatory cytokines by qRT-PCR in endothelial cells 2H-11. We demonstrated that the plasmid construction was successful and can efficiently silence the expression of the two targeted genes. As a consequence of silencing, reduced migration rate and angiogenic potential was confirmed in 2H-11 endothelial cells. Furthermore, induction of DNA sensor STING and proinflammatory cytokines were determined, which could add to the therapeutic effectiveness when used in vivo. To conclude, we successfully constructed a novel plasmid DNA with two shRNAs, which holds a great promise for further in vivo testing.https://www.mdpi.com/1422-0067/22/6/3069CD105CD146plasmidgene electrotransferantibiotic-freeirradiation |
| spellingShingle | Monika Savarin Urska Kamensek Katarina Znidar Vesna Todorovic Gregor Sersa Maja Cemazar Evaluation of a Novel Plasmid for Simultaneous Gene Electrotransfer-Mediated Silencing of CD105 and CD146 in Combination with Irradiation International Journal of Molecular Sciences CD105 CD146 plasmid gene electrotransfer antibiotic-free irradiation |
| title | Evaluation of a Novel Plasmid for Simultaneous Gene Electrotransfer-Mediated Silencing of CD105 and CD146 in Combination with Irradiation |
| title_full | Evaluation of a Novel Plasmid for Simultaneous Gene Electrotransfer-Mediated Silencing of CD105 and CD146 in Combination with Irradiation |
| title_fullStr | Evaluation of a Novel Plasmid for Simultaneous Gene Electrotransfer-Mediated Silencing of CD105 and CD146 in Combination with Irradiation |
| title_full_unstemmed | Evaluation of a Novel Plasmid for Simultaneous Gene Electrotransfer-Mediated Silencing of CD105 and CD146 in Combination with Irradiation |
| title_short | Evaluation of a Novel Plasmid for Simultaneous Gene Electrotransfer-Mediated Silencing of CD105 and CD146 in Combination with Irradiation |
| title_sort | evaluation of a novel plasmid for simultaneous gene electrotransfer mediated silencing of cd105 and cd146 in combination with irradiation |
| topic | CD105 CD146 plasmid gene electrotransfer antibiotic-free irradiation |
| url | https://www.mdpi.com/1422-0067/22/6/3069 |
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