Jing Liu1, Jia-Xin Shen1,2, Guo-Jun Zhang3,4. 1. Chang Jiang Scholar's Laboratory/Guangdong Provincial Key Laboratory for Diagnosis and Treatment of Breast Cancer, Shantou University Medical College, Shantou, Guangdong Province, People's Republic of China. 2. The Breast Center, Cancer Hospital of Shantou University Medical College, 7 Raoping Road, Shantou, Guangdong Province, 515031, People's Republic of China. 3. Chang Jiang Scholar's Laboratory/Guangdong Provincial Key Laboratory for Diagnosis and Treatment of Breast Cancer, Shantou University Medical College, Shantou, Guangdong Province, People's Republic of China. guoj_zhang@yahoo.com. 4. The Breast Center, Cancer Hospital of Shantou University Medical College, 7 Raoping Road, Shantou, Guangdong Province, 515031, People's Republic of China. guoj_zhang@yahoo.com.
Abstract
PURPOSE: Dysregulation of microRNAs (miRNAs) are not only involved in the formation of malignant tumors but also in the processes of differentiation and aggressiveness. However, current methods for detecting miRNA expression have major disadvantages, such as being invasive and non-reproducible. The epithelial-mesenchymal transition (EMT) has been implicated as a pivotal event in the metastasis, stemness, and chemoresistance of malignant tumors. PROCEDURES: In our study, we constructed a new reporter gene, Luc2/tdT_miR200c_3TS, to examine the in vitro and in vivo expression of miR-200c, an EMT-associated miRNA. Quantitative real-time PCR was used to measure the expression levels of miR-200c and EMT-related mRNA, and luciferase assay and bioluminescence imaging were used to measure the luciferase activities in vitro and in vivo, respectively. RESULTS: We found that the expression level of miR-200c was negatively associated with cell migration and invasion. Luciferase activities were regulated by the differential expression levels of miR-200c and EMT process. CONCLUSIONS: Our results demonstrate that Luc2/tdT_miR200c_3TS may be a useful tool for monitoring the expression level of miR-200c at both the cellular level and in living animals, thereby providing a potential high-throughput approach for anticancer drug screening.
PURPOSE: Dysregulation of microRNAs (miRNAs) are not only involved in the formation of malignant tumors but also in the processes of differentiation and aggressiveness. However, current methods for detecting miRNA expression have major disadvantages, such as being invasive and non-reproducible. The epithelial-mesenchymal transition (EMT) has been implicated as a pivotal event in the metastasis, stemness, and chemoresistance of malignant tumors. PROCEDURES: In our study, we constructed a new reporter gene, Luc2/tdT_miR200c_3TS, to examine the in vitro and in vivo expression of miR-200c, an EMT-associated miRNA. Quantitative real-time PCR was used to measure the expression levels of miR-200c and EMT-related mRNA, and luciferase assay and bioluminescence imaging were used to measure the luciferase activities in vitro and in vivo, respectively. RESULTS: We found that the expression level of miR-200c was negatively associated with cell migration and invasion. Luciferase activities were regulated by the differential expression levels of miR-200c and EMT process. CONCLUSIONS: Our results demonstrate that Luc2/tdT_miR200c_3TS may be a useful tool for monitoring the expression level of miR-200c at both the cellular level and in living animals, thereby providing a potential high-throughput approach for anticancer drug screening.
Entities:
Keywords:
Bioluminescence; Breast cancer; In vivo imaging; miR-200c
Authors: Sarah Atef Fahim; Mahmoud Salah Abdullah; Nancy A Espinoza-Sánchez; Hebatallah Hassan; Ayman M Ibrahim; Sarah Hamdy Ahmed; George Shakir; Mohamed A Badawy; Nadia I Zakhary; Burkhard Greve; Mohamed El-Shinawi; Martin Götte; Sherif Abdelaziz Ibrahim Journal: Biomolecules Date: 2020-07-16