Investigating the Functional Significance of a Novel lncRNA in Breast Cancer

Breast cancer is the most common cancer and the main cause of cancer deaths among women globally. Previous research has identified non-coding RNAs (ncRNAs), particularly long ncRNAs (lncRNAs), as key players in cancer development and progression. NcRNAs are RNA molecules that do not possess the ability to encode proteins. This research aims to uncover the functions of RNAI, a novel lncRNA, in breast cancer. RNAI was recently identified as an unannotated region in the genome of MCF7 breast cancer cells, but was absent in the non-cancerous breast MCF10A cells. This project investigates the impact of RNAI knockout/knockdown on breast cancer cell behavior utilizing CRISPR-Cas9 for knockout and small interfering RNAs (siRNAs) for knockdown. Our hypothesis suggests that RNAI knockout/knockdown will modulate breast cancer cell behavior, specifically influencing proliferation, apoptosis, and inflammatory responses depending on the RNAI's potential interactions with the miRNAs hsa-miR-6973-30 and hsa-miR-570-3p that modulate these pathways. Subsequently, after performing RNAI knockout/knockdown in MCF7 cells, assays will be employed to assess cell apoptosis (via Annexin V/Propidium iodide flow cytometry), and inflammatory response (monitoring MyD88 and PD-L1 expression). The results of this project indicates that RNAI is highly expressed in Luminal A subtype of breast cancer suggesting its expression might be subtype specific. Although CRISPR/Cas9 failed to knockout RNAI in MCF7 cells, successful knockdown with siRNAs shows the potential for targeted knockdown of RNAI in cancer cells. Additionally, the success in creating plasmids for CRISPR/Cas9 was confirmed by sequencing, validating their readiness for transfection. The data also indicates that RNAI might have different interactions with different miRNAs and verifies RNAI decoy interaction with hsa-miR-570-3p. Overall, this study offers insights for future therapeutic interventions and investigations into RNAI's precise role.