In the present study, we profiled snoRNA expression signatures of early stage NSCLC by performing microarray analysis on surgical tissues. Aberrant expressions of the identified signatures were well confirmed by RT-qPCR assay. To the best of our knowledge, this is the first study to globally analyze snoRNA expression patterns in human tumor tissues. Furthermore, we demonstrated that like miRNAs, snoRNAs remained intact and were readily detectable in plasma by using RT-qPCR. More importantly, based on the discoveries, we developed a panel of plasma-based snoRNAs as potential biomarkers for early stage NSCLC. Our data might provide compelling evidence that dysregulations of the snoRNAs could play an important role in lung tumorigenesis, and measuring plasma snoRNAs might serves as a potential noninvasive approach to improve diagnosis of NSCLC.
Although rarely being reported, malfunction of some snoRNAs have recently been considered to contribute to carcinogenesis. For instance, adeno-associated viruses integrated their genome into mouse genome, causing liver cancer [
24]. Interestingly, the integration sites identified in the tumors were all located within a DNA interval encoding snoRNAs [
24]. Furthermore, the accumulation of gas5-generated snoRNAs was associated with an arrest of cell growth [
25], and dysregulations of the snoRNAs were related to growth arrest of breast cancer cells [
26,
27]. In addition, although snoRNAs and miRNAs are generated by different cellular pathways and function in different cellular compartments, some members of these two types of ncRNAs display numerous genomic similarities [
28‐
30]. Indeed, a number of human snoRNAs with miRNA-like processing signatures were recently identified [
31]. The findings were consistent with those in another report [
32], in which, a set of miRNAs display functional snoRNA characteristics, and the miRNAs might evolve from snoRNAs [
32]. Therefore, some small ncRNAs in human cells that originate from snoRNAs were proposed to function like miRNAs [
32]. Moreover, snoRNAs could play a role in posttranscriptional gene silencing. For example, HBII-52, a human SNORD gene, can regulate splicing of serotonin receptor 2C messenger RNA [
33]. Like miRNAs, some snoRNAs are located at a chromosomal breakpoint involved in human carcinogenesis. For example, U50 snoRNA was originally discovered from the breakpoint of chromosomal translocation t (3,6) (q27;q15), which was involved in human B-cell lymphoma [
34]. It have been suggested that the genes that are frequently located at chromosomal genomic amplification regions might have oncogenic function involved in the promotion of cancer [
35‐
37]. Notably, all the snoRNAs identified in the present study displayed up-regulation in lung tumor specimens. Interestingly, the snoRNAs are located in commonly frequent genomic amplified regions in lung cancer [
38,
39]. SNORD33 is located in chromosome 19q13.3 that contain potential oncogenes in lung cancer [
36,
37], while SNORD66 and SNORD76 are situated in chromosomal regions 3q27.1 and 1q25.1, respectively. 3q27.1 and 1q25.1 are two of the most frequently amplified chromosomal segments in solid tumors, particularly NSCLC [
35‐
39]. Therefore, upregulation of the snoRNAs in lung cancer might have oncogenic functions in the cacinogensis. Our primary goal of the current study is marker development. The biological relevance of the snoRNAs in tumorigenesis is currently being investigated at our laboratory.
Most of the previously identified lung cancer associated molecular genetic changes were related to the smoking status. Furthermore, some of the changes were associated with lung inflammatory diseases, especially COPD [
40]. The use of such molecular alterations as biomarkers will produce false positive diagnostic rate, thus impeding their future application in clinical settings for diagnosis of lung cancer. The snoRNAs identified from the present research is fairly encouraging as biomarkers, because they highly express in plasma independently of participants' age, gender, ethnic subgroup, and smoking packer-year. In particular, high expressions of SNORD33 and SNORD76 were only observed in plasma from cancer patients. Furthermore, although SNORD66 displayed increased expression in plasma of COPD patients as compared with that in plasma of the healthy controls, it had considerably higher plasma expression level in NSCLC patients compared with COPD individuals. The observation suggests that the snoRNA panel could serves as useful biomarkers in differentiating NSCLC patients from not only healthy individuals, but also COPD subjects. Nonetheless, futures studies to comprehensively investigate biological relevance of the dysregulated SNORD66 in COPD are needed. Moreover, no significant difference regarding plasma expression of the genes was observed at different stages of NSCLC, implying that the potential markers were not stage-specific. In addition, the elevated plasma expression levels of the snoRNAs had equal frequency between AC and SCC of the lungs, suggesting that the genetic changes might be useful biomarkers for the two major histological types of lung cancer.
Although the results look promising, the sensitivity (81.1%) and specificity (95.5%) of the snoRNAs are still not yet efficient for routine clinical application. To surmount the problem, we need to identify additional cancer-associated ncRNAs that can be added to the current ones so that the diagnostic efficacy of the plasma-based approach could be improved. The fundamental mechanism supports this premise is that although only about 352 snoRNAs were analyzed, more than 500 snoRNAs might exist in the human genomic sequences [
41]. Furthermore, we expect to improve such result through combing lung cancer-associated miRNAs with the identified snoRNAs [
11,
13,
14]. In addition, other types of ncRNAs, such as piwi-associated RNAs may also play important role in carcinogenesis [
5,
42]. Including other classes of small ncRNAs with high associated with NSCLC would also improve diagnostic accuracy of the noninvasive approach. To that end, we are analyzing tumor specimens by applying microarray platform to target various types of ncRNAs to develop additional markers for NSCLC.