Upregulation of circular and linear METTL3 and USP3 in colorectal cancer.

Several screening methods are currently used to detect colorectal cancer (CRC). However, these are either under-utilized due to their invasive nature or are limited in terms of their diagnostic ability. Numerous reports have investigated messenger and circular RNA as non-invasive biomarkers, but the majority of gene expression studies using RT-qPCR involve critical errors that often lead to irreproducible findings. In the present study, several of these issues were addressed. To the best of our knowledge, this study reports for the first time the upregulation of both the circular and the linear isoform of USP3 and METTL3 in leukocytes from patients with CRC. The linear transcripts of USP3 and METTL3 exhibited 2.3- and 2-fold increases on average in CRC samples (n=42 CRC) compared with the respective healthy controls (n=32), whereas their circular isoforms showed 1.6- and 1.7-fold increases, respectively. Moreover, a strong positive correlation was observed between the circular and linear isoforms of USP3 in the CRC cohort (P<0.0001), but not in the control group (P>0.05). In addition, the linear USP3 assay had excellent sensitivity (79%), specificity (75%), positive predictive value (81%), negative predictive value (73%) and area under the curve (AUC, 0.8534; P-value <0.0001). The circular (AUC, 0.6946; P-value =0.0043) and linear (AUC, 0.7202; P-value =0.0012) METTL3 assays also showed potential; however, this was not the case for the circular USP3 assay (P-value >0.05). Taken together, this stringent RT-qPCR approach provides evidence for the viability of using circular and linear RNA molecules as disease biomarkers and may help shed light on the regulatory pathways of CRC.