Abstract
Neoadjuvant radiotherapy/chemoradiotherapy (N-RT/CRT) is frequently used to treat rectal cancer due to its ability to reduce tumour bulk prior to surgery. It has been shown to significantly reduce the recurrence of cancer and improve the anal structure integrity post-surgery. However, radiotherapy is often associated with severe adverse effects, drastically reducing the quality of life. Additionally, some patients are subjected to these adverse effects with no positive outcomes as 30% have no response to N-RT/CRT. Therefore, it is essential to understand why tumours respond differently to a treatment. One way to explore this is to look at the molecular characteristics of cancers such as miRNAs involved in regulating gene expression and response to irradiation exposure. This study investigates the expression of miRNAs in response to irradiation exposure in a rectal cancer cell model.
This study used the rectal cancer cell line SW837 to research miRNA as biomarkers of radiosensitivity. Usually, colorectal cancer research is conducted with the faster, more stable colon cancer cell lines. However, we believed researching a rectal cancer cell line would be more representative of rectal cancer because rectal cancers are genetically distinct from colon cancers. The characterisation of irradiation effects on cell viability was important to investigate as our lab had never worked on radiating cells before. SW837 is also relatively underrepresented in research. Therefore, optimisation of all assays was conducted to lay the foundation for future research. After irradiation characterisation, we studied the expression differences in a pre-selected miRNA panel of interest when exposed to different irradiation doses in our SW837 cells.
When characterising the cell viability, death, and apoptosis of SW837 cells exposed to different irradiation doses, a longer incubation post-irradiation incubation time produced the greatest distinction between doses. This was concluded that irradiation-induced cell responses occur the greatest after a full cell cycle has occurred. Therefore, an incubation time of at least 72-hours was ideal as it was found that SW837 cells had a doubling time of 90-hours. Western blotting was used to detect γ-H2AX, a sign of irradiation damage. However, it failed to be detected using western blotting, highlighting the importance of correct protein loading.
miRNA expression changes did not result in any statistically relevant data due to a lack of repeats. However, interesting changes were observed in two of the miRNA. miRNA-185-5p showed a decreased expression with increased irradiation dosing, while miRNA-23a-3p showed the opposite. Overall, though these results were not statistically backed, interesting changes to expression have started to appear, indicating areas to research. Furthermore, this study has optimised many assays to further research in our lab regarding irradiation exposure in cell lines.