Expression of miR-16 is not a suitable reference for analysis of serum microRNAs in melanoma patients

Abstract

Background: The evolving paradigm shift towards the molecular characterization of melanoma has expanded to include studies of microRNA (miRNA) expression. As miR-16 has been utilized as a normalizer in serum-based miRNA studies in several cancers, we evaluated miR-16 expression as a potential reference for normalization of serum miRNA expression in melanoma patients. Methods: 143 primary cutaneous melanoma patients who presented to New York University (NYU) Langone Medical Center for surgical resection of AJCC stage I-III disease were studied. In addition, sera samples from 60 control subjects were utilized including 22 healthy volunteers, 13 rheumatoid arthritis patients, 20 non-melanoma cancer patients (10 renal cell carcinoma and 10 bladder cancer), and 5 Atypical Mole Syndrome patients. The Kruskal-Wallis test (k = 6) or Wilcoxon test (k = 2) with Bonferroni correction was used for analyses of miR-16 expression in melanoma patients compared to various control groups, using raw Ct values directly. The Kruskal-Wallis test was used to compare miR-16 expression across stages of melanoma. The equivalence test for independent samples was used to test the equivalence of miR-16 expression among different groups. Results: No significant differential expression of miR-16 was observed between melanoma patients and healthy volunteers (Wilcoxon test, p = 0.37). However, miR-16 did show a significant difference in expression as it related to stage of melanoma (p = 0.015). Additionally, the equivalence test was unable to confirm equivalent expression of miR-16 in any melanoma versus control group pair. Conclusion: Our data indicate that miR-16 cannot be used as a universal normalizer in sera studies of melanoma patients.