Abstract
BackgroundTo identify aberrant promoter methylation of genomic loci encoding microRNA (mgmiR) in head and neck squamous cell carcinoma (HNSCC) and to evaluate a biomarker panel of mgmiRs to improve the diagnostic accuracy of HNSCC in tissues and saliva.MethodsMethylation of promoter regions of mgmiR candidates was initially screened using HNSCC and control cell lines and further selected using HNSCC and control tissues by quantitative methylation-specific PCR (qMS-PCR). We then examined a panel of seven mgmiRs for validation in an expanded cohort including 189 HNSCC and 92 non-HNSCC controls. Saliva from 86 pre-treatment HNSCC patients and 108 non-HNSCC controls was also examined using this panel of seven mgmiRs to assess the potentials of clinical utilization.ResultsAmong the 315 screened mgmiRs, 12 mgmiRs were significantly increased in HNSCC cell lines compared to control cell lines. Seven out of the 12 mgmiRs, i.e., mgmiR9-1, mgmiR124-1, mgmiR124-2, mgmiR124-3, mgmiR129-2, mgmiR137, and mgmiR148a, were further found to significantly increase in HNSCC tumor tissues compared to control tissues. Using multivariable logistic regression with dichotomized variables, a combination of the seven mgmiRs had sensitivity and specificity of 92.6 and 92.4% in tissues and 76.7 and 86.1% in saliva, respectively. Area under the receiver operating curve for this panel was 0.97 in tissue and 0.93 in saliva. This model was validated by independent bootstrap validation and random forest analysis.ConclusionsmgmiR biomarkers represent a novel and promising screening tool, and the seven-mgmiR panel is able to robustly detect HNSCC in both patient tissue and saliva.
Highlights
To identify aberrant promoter methylation of genomic loci encoding microRNA in head and neck squamous cell carcinoma (HNSCC) and to evaluate a biomarker panel of methylated genomic loci encoding miRNAs (mgmiRs) to improve the diagnostic accuracy of HNSCC in tissues and saliva
DNA methylation at genomic loci encoding microRNAs were identified in HNSCC cell lines We utilized the following approaches to screen candidate methylated genomic loci encoding miRNAs in HNSCC: (1) The UCSC genome browser was used to obtain 1 Kb genomic sequences [GRch38] of 5′-UTRs of 315 primary (Pri-) miRNAs in the Homo sapiens miRBase [from has-let-7a-1 to has-mir-499b in miRbase, http://www.mirbase.org]
(2) The CpG island prediction software (MethPrimer) was utilized to identify 26 genomic loci with CpG islands. (3) By designing methylation-specific primers and running quantitative methylation-specific PCR, we amplified methylation signals in 22 mgmiRs and found 12 mgmiRs that had increased methylation in human HNSCC cell lines compared to normal head and neck cell lines (Additional file 1: Figures S1 and S2)
Summary
To identify aberrant promoter methylation of genomic loci encoding microRNA (mgmiR) in head and neck squamous cell carcinoma (HNSCC) and to evaluate a biomarker panel of mgmiRs to improve the diagnostic accuracy of HNSCC in tissues and saliva. Head and neck squamous cell carcinoma (HNSCC) compromises approximately 90% of all head and neck cancers and 5% of all malignancies [1, 2]. HNSCC has seen an increasing rate of prevalence over the past 30 years due to HPV infection [3]. Despite advancements in cancer therapy, the prognosis for HNSCC patients remains poor [3]. One of the main reasons for the poor prognosis of HNSCC is that by the time of diagnosis, more than half of HNSCC patients have locoregionally advanced disease. Early detection may be key to improving survival rates in the future [4]
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