A Three–MicroRNA Signature as a Potential Biomarker for the Early Detection of Oral Cancer
Abstract
:1. Introduction
2. Results
2.1. Characteristics of Study Subjects
2.2. Expression Profiling of miRNAs by Small RNA-seq
2.3. Selection of Suitable Reference miRNAs
2.4. Investigation of the Six Candidate miRNAs in Training Phase
2.5. Validation of Three Significant miRNA with an Independent Cohort
2.6. Correlations between miRNA Signature and Clinical Parameters
2.7. Logistic Regression Analysis of miRNA Biomarkers
2.8. Diagnostic Performance of miRNA Signature
2.9. Bioinformatics Analysis of miR-222-3p, miR-423-5p, and miR-150-5p
3. Discussion
4. Materials and Methods
4.1. Clinical Samples
4.2. Small RNA Library Preparation and Sequencing
4.3. Small RNA Sequencing Analysis
4.4. miRNA Quantification by qRT-PCR Assays
4.5. Statistical Analysis
4.6. Bioinformatics Analysis
Supplementary Materials
Acknowledgments
Author Contributions
Conflicts of Interest
Abbreviations
OSCC | Oral squamous cell carcinoma |
OL | Oral leukoplakia |
miRNAs | MicroRNAs |
qRT-PCR | Quantitative real-time polymerase chain reaction |
PMD | Potentially malignant disorders |
ROC | Receiver operating characteristic |
AUC | Area under curve |
TCGA | The Cancer Genome Atlas |
Appendix A
mir-222-3p | mir-423-5p | mir-150-5p | ||||
---|---|---|---|---|---|---|
Variables | ρ | p-Value | ρ | p-Value | ρ | p-Value |
Age | 0.105 | NS | 0.037 | NS | −0.052 | NS |
Gender | 0.039 | NS | 0.004 | NS | 0.103 | NS |
Betel chewing status | −0.241 | 0.005 | −0.149 | NS | −0.099 | NS |
Smoking status | −0.161 | NS | 0.034 | NS | 0.001 | NS |
Alcohol status | −0.121 | NS | 0.086 | NS | −0.178 | NS |
Clinical stage | −0.201 | 0.032 | −0.237 | 0.011 | −0.116 | NS |
T stage | −0.220 | NS | −0.276 | 0.003 | −0.156 | NS |
Lymph node metastasis | −0.222 | 0.018 | −0.220 | 0.019 | 0.012 | NS |
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Screening and Training Phase (n = 72) | Validation Phase (n = 178) | |||||
---|---|---|---|---|---|---|
Variables | Normal (%) | OL (%) | OSCC (%) | Normal (%) | OL (%) | OSCC (%) |
Number | 20 | 20 | 32 | 50 | 46 | 82 |
Age (mean ± SD) | 52.05 ± 12.78 | 52.20 ± 12.53 | 52.20 ± 9.03 | 52.86 ± 14.06 | 48.35 ± 12.11 | 53.79 ± 11.25 |
Sex | ||||||
Male | 20 (100.0) | 18 (90.0) | 31 (96.8) | 48 (96.0) | 44 (95.6) | 80 (97.5) |
Female | 0 (0.0) | 2 (10.0) | 1 (3.2) | 2 (4.0) | 2 (4.4) | 2 (2.5) |
Smoking | ||||||
Non-smoker | 4 (20.0) | 6 (30.0) | 1 (3.2) | 2 (4.0) | 6 (13.0) | 7 (8.5) |
Former smoker | 9 (45.0) | 11 (55.0) | 12 (37.5) | 15 (30.0) | 12 (26.1) | 16 (19.5) |
Current smoker | 7 (35.0) | 3 (15.0) | 19 (59.3) | 33 (66.0) | 28 (60.9) | 59 (72.0) |
BQ chewing | ||||||
Non-BQ | 11 (55.0) | 6 (30.0) | 3 (9.4) | 27 (54.0) | 12 (26.1) | 7 (8.5) |
Former BQ chewing | 6 (30.0) | 10 (50.0) | 25 (78.1) | 15 (30.0) | 28 (60.9) | 63 (76.8) |
Current BQ-chewing | 3 (15.0) | 4 (20.0) | 4 (12.5) | 8 (16.0) | 6 (13.0) | 12 (14.7) |
Alcohol consumption | ||||||
Non-drinker | 5 (25.0) | 9 (45.0) | 11 (34.4) | 20 (40.0) | 20 (43.5) | 22 (26.8) |
Former drinker | 10 (50.0) | 9 (45.0) | 13 (40.6) | 22 (44.0) | 23 (50.0) | 36 (43.9) |
Current drinker | 5 (25.0) | 2 (10.0) | 8 (25.0) | 8 (16.0) | 3 (6.5) | 24 (29.3) |
Stage | ||||||
I | 14 (43.8) | 32 (39.0) | ||||
II | 0 (0.0) | 15 (18.3) | ||||
III | 0 (0.0) | 11 (13.4) | ||||
IV | 18 (56.2) | 24 (29.3) | ||||
T stage | ||||||
T1 | 14 (43.8) | 33 (40.2) | ||||
T2 | 3 (9.4) | 24 (29.3) | ||||
T3 | 0 (0.0) | 4 (4.9) | ||||
T4 | 15 (46.8) | 21 (25.6) | ||||
N stage | ||||||
N0 | 19 (59.4) | 60 (73.2) | ||||
N1 | 5 (15.6) | 11 (13.4) | ||||
N2 | 8 (25.0) | 11 (13.4) |
Univariate | Mulitivariate | |||
---|---|---|---|---|
Relative Risk | p-Value | Relative Risk | p-Value | |
Model 1 | ||||
OL | ||||
miR-222-3p | 0.205 (0.123−0.344) | <0.001 | 0.212 (0.127−0.357) | <0.001 |
miR-150-5p | 1.114 (1.027−1.210) | 0.010 | 1.124 (1.032−1.223) | 0.007 |
miR-423-5p | 0.880 (0.673−1.150) | 0.349 | 0.897 (0.682−1.180) | 0.437 |
miR panel a | 1.348 (1.233−1.474) | <0.001 | 1.361 (1.238−1.496) | <0.001 |
OSCC | ||||
miR-222-3p | 1.038 (0.858−1.256) | 0.699 | 1.114 (0.898−1.383) | 0.324 |
miR-150-5p | 1.189 (1.083−1.306) | <0.001 | 1.198 (1.079−1.330) | 0.001 |
miR-423-5p | 1.466 (1.182−1.817) | <0.001 | 1.599 (1.238−2.066) | <0.001 |
miR panel b | 1.377 (1.198−1.584) | <0.001 | 1.386 (1.189−1.615) | <0.001 |
Model 2 | ||||
OSCC | ||||
miR-222-3p | 2.915 (2.087−4.072) | <0.001 | 3.014 (2.102−4.321) | <0.001 |
miR-150-5p | 1.038 (1.003−1.075) | 0.035 | 1.048 (1.007−1.091) | 0.020 |
miR-423-5p | 1.581 (1.238−2.021) | <0.001 | 1.601 (1.236−2.075) | <0.001 |
miR panel c | 1.455 (1.308−1.617) | <0.001 | 1.448 (1.292−1.623) | <0.001 |
Term | No. of Genes | p-Value | Fold Enrichment |
---|---|---|---|
Wnt signaling pathway | 16 | 4.69 × 10−6 | 4.22 |
Pathways in cancer | 26 | 6.07 × 10−5 | 2.41 |
Hepatitis B | 14 | 1.55 × 10−4 | 3.51 |
Axon guidance | 13 | 1.70 × 10−4 | 3.72 |
Sphingolipid signaling pathway | 11 | 1.58 × 10−3 | 3.33 |
HTLV-I infection | 17 | 1.66 × 10−3 | 2.42 |
PI3K-Akt signaling pathway | 20 | 2.71 × 10−3 | 2.11 |
Proteoglycans in cancer | 14 | 3.19 × 10−3 | 2.55 |
FoxO signaling pathway | 11 | 3.57 × 10−3 | 2.99 |
Rap1 signaling pathway | 14 | 4.79 × 10−3 | 2.42 |
MicroRNAs in cancer | 17 | 4.84 × 10−3 | 2.17 |
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Chang, Y.-A.; Weng, S.-L.; Yang, S.-F.; Chou, C.-H.; Huang, W.-C.; Tu, S.-J.; Chang, T.-H.; Huang, C.-N.; Jong, Y.-J.; Huang, H.-D. A Three–MicroRNA Signature as a Potential Biomarker for the Early Detection of Oral Cancer. Int. J. Mol. Sci. 2018, 19, 758. https://doi.org/10.3390/ijms19030758
Chang Y-A, Weng S-L, Yang S-F, Chou C-H, Huang W-C, Tu S-J, Chang T-H, Huang C-N, Jong Y-J, Huang H-D. A Three–MicroRNA Signature as a Potential Biomarker for the Early Detection of Oral Cancer. International Journal of Molecular Sciences. 2018; 19(3):758. https://doi.org/10.3390/ijms19030758
Chicago/Turabian StyleChang, Yi-An, Shun-Long Weng, Shun-Fa Yang, Chih-Hung Chou, Wei-Chih Huang, Siang-Jyun Tu, Tzu-Hao Chang, Chien-Ning Huang, Yuh-Jyh Jong, and Hsien-Da Huang. 2018. "A Three–MicroRNA Signature as a Potential Biomarker for the Early Detection of Oral Cancer" International Journal of Molecular Sciences 19, no. 3: 758. https://doi.org/10.3390/ijms19030758