Ravinderan, Presela (2021) Generation Of RNA Aptamer Against Progesterone Receptor DNA Binding Domain And Its Potential In Diagnostic And Therapeutic Applications In Breast Cancer. Masters thesis, Universiti Sains Malaysia.
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Abstract
Aptamers are a new class of molecular recognition element that exhibit high binding affinity and specificity against the target. In this study, an RNA aptamer was generated against Progesterone Receptor DNA binding domain (PR DBD) that acts as a diagnostic biomarker and potential therapeutic target for breast cancer. Firstly, PR DBD gene was amplified and isolated from the total RNA of MCF-7 cells and cloned into pET15b plasmid. Protein expression and native purification was performed using E. coli Rosetta 2(DE3)pLysS bacterial strain. The identity of the protein was confirmed by western blot using polyclonal anti-PR antibody and by protein sequencing using MALDI-TOF/TOF mass spectrometry. The purified protein was subjected to SELEX in order to isolate RNA aptamer. A total of eight SELEX cycles were executed and the resulting nucleic acid pool from cycle 8 showed enrichment against PR DBD. The cycle 8 nucleic acid pool was sequenced using direct sequencing and crush and soak elution-based sequencing methods. Both sequencing methods revealed the presence of three different classes of sequences, with one class termed, PRapt-3 showed the strongest binding against PR DBD. The dissociation constant of PRapt-3 RNA aptamer was estimated at 380 nM ± 35 nM. PRapt-3 was successfully used to develop aptamer-based diagnostic assays such as ALISA, aptamer-based dot blot, aptamer-based western blot, aptacytostaining and aptahistostaining. PRapt-3 detected PR DBD in direct ALISA with a LOD of 69.44 nM. In the aptamer-based dot blot assay, PRapt-3 detected up to 6.25 pmol of PR DBD. PRapt-3 also successfully detected recombinant PR DBD and endogenous PR DBD in MCF-7 and HeLa cells on aptamer-based western blot assay. PRapt-3 demonstrated nuclear staining in aptacytostaining and with better penetration in aptahistostaining using the formalin fixed paraffin embedded breast cancer cell and tissue blocks. Apart from diagnostic application, the functionality of the PRapt-3 RNA aptamer was also investigated in the therapeutic applications. First, the antagonistic property of the PRapt-3 was investigated on the cell cycle analysis. Flow cytometry analysis showed that PRapt-3 RNA reduced the number of cells in S phase of MCF-7 cell cycle while reciprocally increased the percentage of cells in G0/G1. PRapt-3 can also act as a promising apoptotic inducing agent. In this study, it was found that PRapt-3 downregulated the anti-apoptotic gene, BCL-2 and upregulated the pro-apoptotic gene, BAX. PRapt-3 mitigated expression of the proliferationrelated genes. The expression level of proliferation-related genes, PI3K and AKT were reduced by PRapt-3 RNA. The downregulation of these genes showed that PRapt-3 was successfully employed to antagonize the interaction between PR and progesterone. In this entirety, PRapt-3 is a promising diagnostic and therapeutic agent.
| Item Type: | Thesis (Masters) |
|---|---|
| Subjects: | R Medicine |
| Divisions: | Institut Perubatan & Pergigian Termaju (Advanced Medical & Dental Institute (AMDI)) > Thesis |
| Depositing User: | NOR HASHIMY BIN ABDULLAH |
| Date Deposited: | 22 Apr 2026 02:27 |
| Last Modified: | 22 Apr 2026 02:27 |
| URI: | http://eprints.usm.my/id/eprint/63950 |
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