Publication: Reseptör tabanlı aptamerin apoptotik etkisinin meme kanseri hücre hatlarında miRNA profillenmesi
Abstract
Meme kanseri (BC), dünya genelinde kadınlar arasında en sık tanı konulan malignitedir, kansere bağlı ölüm nedenleri arasında ikinci sırada yer almaktadır. Epidermal Büyüme Faktörü Reseptörünün (EGFR) aşırı ekspresyonu özellikle Üçlü Negatif Meme Kanserinde (TNBC) karakteristik bir durumdur. EGFR reseptörünün hedef alınarak geliştirilmeyi amaçlandığı moleküler tedavi stratejileri günümüzde önem kazanmıştır. Aptamerler, hedef moleküllere karşı yüksek özgüllük ve bağlanma afinitesi gösteren, kısa tek sarmallı DNA (ssDNA) veya RNA yapısında oligonükleotidlerdir. Epidermal Büyüme Faktörü Reseptörünü hedefleyen ssDNA tabanlı aptamerlerin, kanser hücrelerinin proliferasyonunu ve sağkalımını negatif yönde etkileyebileceği düşünülmektedir. Bunun yanı sıra, kısa ve kodlamayan RNA’lar olan mikroRNA’ların (miRNA), hücresel sinyal yolaklarının regülasyonunda önemli rol oynadığı ve karsinogenez sürecine katkıda bulunduğu çeşitli çalışmalarda gösterilmiştir. Bu bağlamda, TNBC doku örneklerinde miR-181b-5p'nin anlamlı düzeyde artış gösterdiği; bu artışın da kötü prognoz ve düşük sağkalım oranlarıyla ilişkili olduğu rapor edilmiştir. Bu çalışmada, EGFR’ye özgü ssDNA aptamerinin (TuTu22), MDA-MB-231 meme kanseri hücre hattı üzerindeki antiproliferatif ve apoptotik etkileri çok yönlü olarak değerlendirilmiştir. Uygulanan MTT hücre canlılık analizleri sonucunda, aptamerin 200 nM konsantrasyonunda ve 72 saatlik inkübasyon süresinde, doza ve zamana bağlı olarak hücre canlılığında anlamlı azalmaya yol açtığı tespit edilmiştir. Özellikle 200 nM konsantrasyonda uygulanan EGFR aptameri, MDA-MB-231 hücrelerinde koloni oluşumunu ve hücre göçünü baskılayarak belirgin bir sitostatik etki oluşturmuştur. Aynı zamanda, aptamer uygulaması sonucunda mitokondriyal membran potansiyelinde azalma, reaktif oksijen türlerinin (ROS) artışı ve hücre ölümünde artış gözlemlenmiştir. qRT-PCR ile gerçekleştirilen miRNA ekspresyon analizinde ise EGFR aptamerine maruz kalan MDA-MB-231 hücrelerinde miR-21-5p, miR-181b-5p ve miR-155-5p düzeylerinin sırasıyla 5, 7 ve 1.2 kat arttığı belirlenmiştir. Bu bulgular, Epidermal Büyüme Faktörü Reseptörüne özgü aptamerin miRNA ekspresyon profillerini anlamlı biçimde modüle ettiğini göstermektedir. Ayrıca, 200 nM EGFR aptamer uygulaması sonrası anti-apoptotik protein olan Bcl-xL ile birlikte kaspaz-3 ve PARP proteinlerinin bölünmesinde de anlamlı düzeyde azalma tespit edilmiştir. Sonuç olarak, EGFR’ye özgü ssDNA aptameri, MDA-MB-231 TNBC hücre hattında hem hücre canlılığını baskılayarak hem de miRNA ekspresyon profillerini değiştirerek apoptotik süreci tetiklemiştir. Bu veriler, EGFR aptamerlerinin, özellikle tedaviye dirençli meme kanseri alt tiplerinde yeni nesil hedefe yönelik terapötik ajanlar olarak potansiyel taşıdığını göstermektedir.
Breast cancer (BC) is the most frequently diagnosed malignancy among women worldwide and ranks as the second leading cause of cancer-related mortality. Overexpression of the Epidermal Growth Factor Receptor (EGFR) is particularly characteristic of Triple-Negative Breast Cancer (TNBC). Targeting EGFR has become increasingly significant in the development of novel molecular therapy strategies. Aptamers are short, single-stranded DNA (ssDNA) or RNA oligonucleotides that exhibit high specificity and binding affinity toward their target molecules. ssDNA-based aptamers targeting the Epidermal Growth Factor Receptor are thought to negatively affect cancer cell proliferation and survival. Additionally, microRNAs (miRNAs), which are short, non coding RNA molecules, play a crucial role in the regulation of cellular signaling pathways and have been shown to contribute to carcinogenesis. In this context, significantly increased levels of miR-181b-5p have been reported in TNBC tissue samples and are associated with poor prognosis and reduced survival rates. In this study, the antiproliferative and apoptotic effects of an EGFR-specific ssDNA aptamer (TuTu22) on the MDA-MB-231 breast cancer cell line were evaluated from multiple perspectives. According to MTT cell viability assays, the aptamer induced a significant decrease in cell viability in a dose- and time-dependent manner, particularly at 200 nM concentration after 72 hours of incubation. At this concentration, the EGFR aptamer notably suppressed colony formation and cell migration in MDA-MB-231 cells, demonstrating a distinct cytostatic effect. Moreover, treatment with the aptamer resulted in mitochondrial membrane depolarization, increased production of reactive oxygen species (ROS), and elevated levels of cell death. qRT-PCR-based miRNA expression analysis revealed that exposure to the EGFR aptamer led to increased expression levels of miR-21-5p and miR-181b-5p (by 5-and 7-fold, respectively), while miR-155-5p levels decreased by approximately 1.2-fold. These findings indicate that the EGFR-specific aptamer significantly modulates miRNA expression profiles. Additionally, treatment with 200 nM EGFR aptamer caused a notable reduction in the anti-apoptotic protein Bcl-xL, along with cleavage of caspase-3 and PARP proteins. In conclusion, the EGFR-specific ssDNA aptamer induced apoptosis in the MDA-MB-231 TNBC cell line by suppressing cell viability and altering miRNA expression profiles. These findings suggest that EGFR aptamers hold potential as next-generation targeted therapeutic agents, particularly in treatment-resistant subtypes of breast cancer.
Breast cancer (BC) is the most frequently diagnosed malignancy among women worldwide and ranks as the second leading cause of cancer-related mortality. Overexpression of the Epidermal Growth Factor Receptor (EGFR) is particularly characteristic of Triple-Negative Breast Cancer (TNBC). Targeting EGFR has become increasingly significant in the development of novel molecular therapy strategies. Aptamers are short, single-stranded DNA (ssDNA) or RNA oligonucleotides that exhibit high specificity and binding affinity toward their target molecules. ssDNA-based aptamers targeting the Epidermal Growth Factor Receptor are thought to negatively affect cancer cell proliferation and survival. Additionally, microRNAs (miRNAs), which are short, non coding RNA molecules, play a crucial role in the regulation of cellular signaling pathways and have been shown to contribute to carcinogenesis. In this context, significantly increased levels of miR-181b-5p have been reported in TNBC tissue samples and are associated with poor prognosis and reduced survival rates. In this study, the antiproliferative and apoptotic effects of an EGFR-specific ssDNA aptamer (TuTu22) on the MDA-MB-231 breast cancer cell line were evaluated from multiple perspectives. According to MTT cell viability assays, the aptamer induced a significant decrease in cell viability in a dose- and time-dependent manner, particularly at 200 nM concentration after 72 hours of incubation. At this concentration, the EGFR aptamer notably suppressed colony formation and cell migration in MDA-MB-231 cells, demonstrating a distinct cytostatic effect. Moreover, treatment with the aptamer resulted in mitochondrial membrane depolarization, increased production of reactive oxygen species (ROS), and elevated levels of cell death. qRT-PCR-based miRNA expression analysis revealed that exposure to the EGFR aptamer led to increased expression levels of miR-21-5p and miR-181b-5p (by 5-and 7-fold, respectively), while miR-155-5p levels decreased by approximately 1.2-fold. These findings indicate that the EGFR-specific aptamer significantly modulates miRNA expression profiles. Additionally, treatment with 200 nM EGFR aptamer caused a notable reduction in the anti-apoptotic protein Bcl-xL, along with cleavage of caspase-3 and PARP proteins. In conclusion, the EGFR-specific ssDNA aptamer induced apoptosis in the MDA-MB-231 TNBC cell line by suppressing cell viability and altering miRNA expression profiles. These findings suggest that EGFR aptamers hold potential as next-generation targeted therapeutic agents, particularly in treatment-resistant subtypes of breast cancer.