Serum long non‐coding RNA, snoRNA host gene 5 level as a new tumor marker of malignant melanoma

Malignant melanoma (MM) is a progressive disease. It is important to detect MM in the early stage using sensitive markers; however, no broadly used tumor markers for early-stage MM have been established. Quantifying cell-free serum RNA is feasible 1, and we previously reported the efficacy of the serum and hair shaft levels of microRNA-221 (miR-221) as melanoma markers 2, 3. However, the ability to detect MM using serum markers continues to be insufficient. Long non-coding RNAs (lncRNAs) are novel regulators of cancer biology 4. Because it is located in inclusion bodies, lncRNA may exist outside of cells. lncRNA is reported as a marker of hepatocellular carcinoma and bladder cancer 5, 6. Therefore, we examined the serum levels of lncRNA as a new tumor marker of MM. We investigated the serum levels of lncRNAs in patients with MM and evaluated the possibility that the serum SNHG5 level may be a useful marker for MM. Serum samples were collected from 24 patients with MM (Table S1). Additionally, we collected serum samples from five patients treated with surgery who later experienced recurrence (Table S2). We also collected serum samples from 15 normal subjects (NS) and five patients with squamous cell carcinoma (SCC) (Table S3) as well as tissue samples from 36 patients with MM and four nevi. Institutional review board approval with written informed consent was obtained before patients and normal volunteers were enrolled in this study according to the Declaration of Helsinki. The samples were analysed using real-time PCR with specific primers for lncRNAs and miR-221. The detailed methodologies are described in the Data S1. We first examined three mixed serum samples of melanoma and three serum samples from the normal subjects, respectively. The samples were analysed using real-time PCR with eight specific primers for lncRNAs (SNHG1, SNHG3, SNHG5, SNHG6, MEG3, MEG9, NEAT1 and MIAT). As a result, only the serum levels of SNHG5 were found to be significantly higher in the patients with melanoma than in the normal subjects (data not shown). Therefore, we decided to examine the SNHG5 levels in further experiments. The serum SNHG5 levels in all MM cases, all MM stages, SCC and NS samples are shown in Fig. 1. The serum levels of SNHG5 were significantly higher in the patients with MM than in the normal subjects (P < 0.001) and patients with SCC (P < 0.05). The serum levels of SNHG5 for each MM stage were significantly higher than those in the NS, especially for stage 4 (stages 0–3, P < 0.05; stage 4, P < 0.01). The ROC curve for the expression of SNHG5 to distinguish patients with melanoma from the normal controls is shown in Fig. 2a. The AUC was 0.86; therefore, we considered SNHG5 to be a useful biomarker of MM. The results of the comparison of the SNHG5 and 5-S-CD levels as biomarkers are shown in Fig. 2b. The 5-S-CD level is commonly used as a MM marker in Japan. Compared with that observed for the 5-S-CD level, the positive rate for the SNHG5 level in the early stages (stages 0–3) was higher. The positive rates of SNHG5 ranged from 60 to 70% at every stage. Figure S1 shows the results of a longitudinal study of the serum levels of SNHG5 in five patients with melanoma. The serum levels of SNHG5 significantly decreased after the surgery (P < 0.05). In addition, in two of the five patients, the serum levels increased again at the time of recurrence. The tissue levels of SNHG5 in the patients with MM and nevus samples are shown in Figure S2. There were no significant differences in the levels of SNHG5 between the MM tissue and nevus samples (P = 0.14). The main producer of SNHG5, tumor or normal cells, in patients with cancer remains unknown. We herein found the serum levels of SNHG5 to be significantly upregulated in the patients with melanoma, but they did not increase according to the stage as opposed to the levels of protein tumor markers, such as 5-S-CD. We speculate that the serum SNHG5 level reflects the tumor-bearing status of the patient, but not the progression of cancer. Additionally, we compared SNHG5 with microRNA-221, which we previously reported to be a sensitive marker (Figure S3 and Table S1). We also examined the half-life of SNHG5 in the serum (Figure S4). SNHG5 is a member of the non-coding multiple small nucleolar RNA host gene family. The SNHG5 gene is 524 bp in size and located on chromosome 6q15 at the breakpoint of chromosomal translocation t3, 6(q27;q15); this site is involved in human B-cell lymphoma 7. The fact that the serum levels of SNHG5 were upregulated in patients with MM suggests that SNHG5 may play some roles in melanomagenesis and/or melanoma metastasis. In this study, the serum levels of SNHG5 did not increase in the patients with SCC. However, the present results are associated with some limitations due to the small number of patients. To answer the question as to whether SNHG5 is specifically involved in the pathogenesis of MM, further studies using more samples from other cancers are needed. This work was supported by JSPS KAKENHI grant numbers 25870545 and 15K09772. Y. I. performed the experiments, analysed the data and wrote the manuscript. S. F. designed the research and wrote the manuscript. A. T., J. Y., A. M. and S. N. contributed to the sampling. W.Z., J. A., I. K. and S. M. performed the experiments. M. J. and H. I. provided excellent scientific suggestion. The authors declare no conflict of interests. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. 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