Colorectal cancer (CRC) is the second highest cause of cancer deaths in New Zealand and we have some of the highest rates of CRC worldwide. Diagnosis and treatment while the disease is still in its early stages is key for ensuring the best possible outcome. Some small RNAs, such as piwi-interacting RNAs (piRNA) and microRNAs (miRNA), are dysregulated in tumour tissue and can be exported from tumour cells via EVs where they can act as liquid biopsy markers. This study has investigated small RNA expression in EVs from a cohort of CRC patients to determine whether they can act as liquid biopsy markers for the detection of early stage disease.
Blood samples were obtained from patients at Wellington Regional Hospital prior to any treatment. Controls were matched for age and sex and were confirmed as having a normal bowel, with no polyps, by colonoscopy. Tumour and normal mucosal tissue was collected immediately after bowel resection. EVs were isolated from plasma by qEV size exclusion columns. EVs were imaged by cryo-SEM, protein analysis was conducted by Western blotting and RNA analysis performed by qRT-PCR.
Western blotting confirmed the presence of EVs in the plasma samples through the EV-associated markers CD9 and CD63 and cryo-SEM showed spherical vesicles ranging from 40-300 nm in size. In tissue, piR-1245, miR-183 and miR-1246 were elevated in the tumour tissue of early stage CRC patients when compared to their normal mucosa (n=17 p<0.0001, n=21 p=0.0007, n=37 p=0.0007 respectively, paired t-test). In plasma, piR-1245 was elevated in early stage CRC patients compared to controls (control n=19, early stage n= 20, p=0.011, unpaired t-test). miR-19 expression in plasma EVs was decreased in early stage CRC compared to controls (control n=26, early stage n=62, p=0.019, unpaired t-test).
Circulating EVs in early stage CRC patients have a small RNA profile distinct from that of healthy controls. Interestingly, there is some discrepancy in small RNA dysregulation between tumour tissue and plasma EVs. piR-1245 and miR-19 have potential as blood-based biomarkers that could be used to diagnose early stage CRC.