UTILIZATION OF DOTAP-ENCAPSULATED MIR-204 TO INHIBIT PIK3CB AND CCND1 MRNA ON MCF-7 CANCER CELL LINE

• Main Author: Naura Safira Rizam, Bilqis
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/84680
• Institution: Institut Teknologi Bandung

Drug resistance is the major cause of death of breast cancer patients, with the most common resistance mechanism involving the MDM2 protein that inhibits p53. In order to overcome this drug resistance problem, new treatments are needed to inhibit several signaling pathways including signaling pathway of MDM2. MiR-204 is predicted to target PIK3CB and CCND1 mRNAs that play a role in the upstream and downstream signaling pathways of PI3K/AKT. In this research, miR-204 is encapsulated with DOTAP (1,2-dioleoyl-3-trimethylammonium propane) as a delivery system to avoid nuclease degradation. Therefore, the purpose of this study was to determine the effect of DOTAP-encapsulated miR-204 on breast cancer cell lines (MCF-7) survival and mRNA levels of PIK3CB and CCND1. Morphology and diameter of DOTAP-encapsulated miR-204 nanoparticle is characterized by Transmission Electron Microscopy (TEM) and Particle Size Analyzer (PSA), respectively. Cytotoxicity of DOTAP was observed using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The cytotoxicity of miR-204 encapsulated with DOTAP was also tested and compared with empty DOTAP and Paclitaxel. Internalization of miR-204 was observed at 24 and 48 hours post-incubation. PIK3CB and CCND1 mRNA levels were analyzed using the RT-qPCR method. DOTAP nanoparticle has a spherical shape and the diameter around 37,84 – 295,3 nm, while the diameter of DOTAP-encapsulated miR-204 was 151,57 – 454,69 nm. The morphology and diameter of these nanoparticles are in accordance with the ideal criteria for a delivery system. MTT assay result showed the concentration of DOTAP that can be used in this research is 20 ?g/mL. The viability of MCF-7 cells treated with miR-204 encapsulated with DOTAP was significantly lower (p<0.05) than cells treated with empty DOTAP and was not significantly different (p>0.05) than cells treated with Paclitaxel. DOTAP-encapsulated miR-204 could be successfully internalized by MCF-7 cancer cells at 24 hours post-incubation, with an increased number of dead cells observed after 48 hours of treatment. Furthermore, from RT-qPCR analysis, it was obtained that the levels of PIK3CB and CCND1 mRNA decreased significantly (p<0.05) in cells treated with DOTAP-encapsulated miR-204 compared to empty DOTAP and was not significantly different compared to Paclitaxel (p>0.05). In conclusion, DOTAP-encapsulated miR-204 is able to reduce cell survival and reduce PIK3CB and CCND1 mRNA in MCF-7 cancer cell lines.