Publication: Tenascin-C-CRISPR/ Cas9 plasmidi taşıyan oksitosinle hedeflendirilmiş pegli kitozan nanoplekslerinin meme tümör modelinde tümör büyümesi ve yayılmasına etkisi
Abstract
Amaç: Üçlü negatif meme kanseri en agresif meme kanseri alt tipidir. Tenascin-C bu kanserde çoğalma, metastaz ve anjiyogenezi destekleyen tümöre özgül bir matriks proteinidir. Bu çalışmada, Tenascin-C ekspresyonu CRISPR/ Cas9 genom düzenleme sistemi ile üçlü negatif meme kanserinde azaltılmasını etkilerinin in vitro ve in vivo araştırılması ve güvenli ve etkin in vivo uygulama için oksitosinle hedeflendirilmiş yeni bir kitozan taşıyıcı sistemi formüle edilmesi amaçlanmıştır. Gereç ve Yöntem: İnsan meme kanseri hücre hattı MDA-MB-231 hücreleri, Tenascin-C’yi hedefleyen CRISPR/ Cas9 plazmidleri ile transfekte edildikten sonra ekspresyonundaki düşüş incelenmiştir. Azalan ekspresyonunun hücrelerdeki apoptoz, proliferasyon, göç ve invazyon üzerine etkileri in vitro analiz edilmiştir. PEG bağlayıcı kullanılarak, oksitosin kitozana bağlanmıştır ve CRISPR/ Cas9 plazmidleri ile hazırlanan nanoplekslerin karakterizasyonu yapılmış ve transfeksiyon etkinliği araştırılmıştır. Daha sonra ksenograft meme kanseri fare modelinde biyodağılım ve nanopleksle tedavinin tümöre etkisi analiz edilmiştir. Bulgular: CRISPR/ Cas9 plazmidleri Tenascin-C mRNA ekspresyonunun in vitro çalışmada %60 oranında azaltmış ve hücrelerde apoptoz artarken, proliferasyon, göç ve invazyon kapasitesi düşmüştür. Formüle edilen nanopleksler hücreleri başarıyla transfekte etmiştir fakat oksitosinin damar içi uygulamada tümörü hedeflendirmek için yeterli olmadığı görülmüştür. Nanopleksler intratümöral uygulandığında ise, tümör boyutu kontrol gruplarına göre anlamlı derecede küçülmüştür. Sonuç: Özgün bir kitozan bazlı taşıyıcı sistem formüle edilmiştir ve CRISPR/ Cas9 ile Tenascin-C düzenlemesinin ileri çalışmalarda test edilmesi için temel oluşturulmuştur.
Objective: Triple negative breast cancer is the most aggressive breast cancer subtype. Tenascin-C is a tumor-specific matrix protein that promotes proliferation, metastasis, and angiogenesis in this cancer. In this study, the aims were to investigate the in vitro and in vivo effects of reducing Tenascin-C expression in triple negative breast cancer by the CRISPR/ Cas9 genome editing system, and to formulate a new oxytocin-targeted chitosan delivery system for safe and effective in vivo application. Materials and Methods: The decrease in Tenascin-C expression was investigated after human breast cancer cell line, MDA-MB-231 cells were transfected with CRISPR/ Cas 9 plasmids targeting Tenascin-C. The effects of decreased expression on apoptosis, proliferation, migration and invasion in cells were analyzed in vitro. Using a PEG linker, oxytocin was conjugated to chitosan and the nanoplexes prepared with CRISPR/ Cas9 plasmids were characterized and transfection efficiency was investigated. Then, biodistribution and the effect of nanoplex treatment on tumor were analyzed in a xenograft breast cancer mouse model. Results: CRISPR/ Cas9 plasmids decreased Tenascin-C mRNA expression by 60% and increased apoptosis in cells, while decreasing proliferation, migration and invasion capacity in vitro. Nanoplexes successfully transfected cells, but oxytocin was not sufficient for active tumor targeting in intravenous administration. When nanoplexes were applied intratumorally, tumor sizes were significantly reduced compared to the control groups. Conclusion: A new chitosan-based carrier system has been formulated and foundations have been laid for testing Tenascin-C editing by CRISPR/ Cas9 in the future studies
Objective: Triple negative breast cancer is the most aggressive breast cancer subtype. Tenascin-C is a tumor-specific matrix protein that promotes proliferation, metastasis, and angiogenesis in this cancer. In this study, the aims were to investigate the in vitro and in vivo effects of reducing Tenascin-C expression in triple negative breast cancer by the CRISPR/ Cas9 genome editing system, and to formulate a new oxytocin-targeted chitosan delivery system for safe and effective in vivo application. Materials and Methods: The decrease in Tenascin-C expression was investigated after human breast cancer cell line, MDA-MB-231 cells were transfected with CRISPR/ Cas 9 plasmids targeting Tenascin-C. The effects of decreased expression on apoptosis, proliferation, migration and invasion in cells were analyzed in vitro. Using a PEG linker, oxytocin was conjugated to chitosan and the nanoplexes prepared with CRISPR/ Cas9 plasmids were characterized and transfection efficiency was investigated. Then, biodistribution and the effect of nanoplex treatment on tumor were analyzed in a xenograft breast cancer mouse model. Results: CRISPR/ Cas9 plasmids decreased Tenascin-C mRNA expression by 60% and increased apoptosis in cells, while decreasing proliferation, migration and invasion capacity in vitro. Nanoplexes successfully transfected cells, but oxytocin was not sufficient for active tumor targeting in intravenous administration. When nanoplexes were applied intratumorally, tumor sizes were significantly reduced compared to the control groups. Conclusion: A new chitosan-based carrier system has been formulated and foundations have been laid for testing Tenascin-C editing by CRISPR/ Cas9 in the future studies