Abstract
Colorectal cancer (CRC) has been ranked among the leading cause of cancer-related mortality, accounting for 19.5% of cancer incidence, globally. Over the years, while there have been advancements with therapeutic treatments to improve cancer management, CRC burden remains high especially for metastatic CRC patients. In recent years, extensive research has discovered many cancer-associated mutations within non-coding regions of our genome, including long non-coding RNAs (lncRNAs). LncRNAs are classified as transcripts with >200 nucleotides and do not encode for a protein, however, still have regulatory functions. They have emerged as potential therapeutic targets with a majority being identified as key players in the development and progression of cancer hallmarks. While there are more than 100,000 lncRNA transcripts that have been identified to date, many remain largely uncharacterized.
Therefore, in this study, we sought to characterize a novel lncRNA, termed CAN3, and its role in CRC progression. LncRNA CAN3 was initially identified as a regulator of cell growth in a CRISPR-interference (CRISPRi) depletion screen in a CRC cell line. From this, we hypothesized that CAN3 plays a role in driving cell proliferation in CRC. We first aimed to generate a loss-of-function model in the HCT116 (human colorectal carcinoma) cell line using the CRISPRi approach. Validation of expression levels showed successful CAN3 repression with >50% knockdown efficiency. Our second aim was to generate a loss-of-function model in the HCT116 cell line using antisense oligonucleotides (ASOs) as a means of comparing phenotypical changes between a transcriptional loss-of-function model (CRISPRi) and a post-transcriptional silencing model (ASOs). Similarly, the loss-of-function models generated using ASOs resulted in >50% knockdown efficiencies. The third aim of this study was to elucidate the role of CAN3 on cell proliferation in CRC using the loss-of-function models that had been generated. Our findings showed no significant changes to the rate of cell proliferation upon CAN3 repression, thus, rejecting the hypothesis that was initially proposed.
Taken together, our success with generating knockdown models using CRISPRi and ASOs suggest that both approaches are useful tools for generating loss-of-function models targeting lncRNA genes. Findings from our interrogation of CAN3 suggest that it does not play a role in driving cell proliferation in CRC. However, we propose that further investigations into validating the results observed and mitigating any limitations from this project may be beneficial to further evaluate its potential as a therapeutic target for CRC treatment.