br Materials and Methods br Results br

Materials and Methods
Results
Discussion In this study, using a series of logical and comprehensive approaches, we demonstrated functional oncogenic relevance of SNORA21 in CRC. We made several key observations during this study: First, we observed that expression of SNORA21 is upregulated in CRC using multiple publicly available datasets and we demonstrated in clinical specimens that SNORA21 was even overexpressed in colorectal adenomas. Second, while evaluating the clinical significance of SNORA21 in two independent cohorts of tumor specimens from patients with CRC and observed significant associations between high SNORA21 expression with invasion, metastasis, and tumor progression in patients with CRC. Third, we noted that high SNORA21 expression emerged as an independent predictor of poor overall survival in CRC patients. Fourth, in a series of in vitro and in vivo experiments, we observed that CRISPR-mediated inhibition of SNORA21 resulted in decreased cell proliferation and the inhibited ability for invasion and metastasis, which corroborated with our findings in patient cohorts. One of the major findings of the current study is the oncogenic role of SNORA21 in CRC. SNORA21, comprising of 133 nucleotides and located between exons 2 and 3 of the RPL23 gene, was originally thought to induce rRNA maturation by guiding the psuedouridylation of residues U4401 and U4480 of the 28S rRNA (Ofengand and Bakin, 1997). However, in the present study, through suppression of SNORA21 expression using the CRISPR/Cas9 system, we provided evidence for its oncogenic role in CRC. Results from our study elucidated that SNORA21 could regulate 69 8 with resultant changes in cellular proliferation and tumor invasion. These results were subsequently validated and were further supported by in vivo experiments. To further clarify the oncogenic role of SNORA21, we conducted microarray analysis in CRC cells. Intriguingly, we identified that SNORA21 is involved in several cancer related signaling pathways, including Hippo signaling pathway (Gregorieff et al., 2015), Wnt signal pathway (Fevr et al., 2007) and Axon guidance pathway (Li et al., 2009). Collectively, these data highlights the importance of SNORA21 in regulating key signaling pathways of CRC. Intriguingly, the oncogenic function of SNORA21 also associated with key clinicopathological features in CRC patients. Previously, we have demonstrated prognostic potential of another snoRNA, SNORA42 (Okugawa et al., 2017). We identified SNORA42 based on published reports from other cancers and our discovery process for CRC-related snoRNA candidates was limited. However, in the present study, we used multiple high-throughput RNA expression profiling datasets to identify potential oncogenic snoRNAs in CRC in an unbiased manner. Based upon such a comprehensive discovery strategy we identified that SNORA21 expression was elevated in CRC and adenomas compared to normal colorectal tissues, and its enhanced expression associated with increased invasion and metastasis. Especially, high levels of SNORA21 in adenoma indicate the possibility that it could facilitate development of CRC, and its expression level could also serve as useful detection biomarkers for CRC. Furthermore, we observed that in primary CRCs with low levels of SNORA21 expression, when these tumors metastasized to the liver, levels of SNORA21 were significantly elevated at the metastatic sites – providing a potential causal link for these observations and the suggestion that it may play an important role in disease progression as well. Moreover, we found that SNORA21 expression was higher in primary cancers from CRC patients with multiple metastatic sites compared to those with no metastasis, suggesting that high SNORA21 expression in the primary cancer may drive cell proliferation and cell invasion ability, and eventual metastasis. Therefore, these findings suggest the clinical utility of SNORA21, and potentially other snoRNA-based prognostic and predictive markers for distant metastasis in CRC. In particular, some of the unique characteristics of snoRNAs make them very attractive for use as biomarkers. Due to their localization within the cell, snoRNAs generally are not affected by hemolysis and are relatively stable in blood by binding to as yet unidentified proteins (Zhang et al., 2012). Collectively, the results of our study highlights the potential of snoRNAs to be used as diagnostic, prognostic markers, and may perform similarly to miRNAs as non-invasive biomarkers (Toiyama et al., 2013, 2014).