Plasma miRNA-146b-3p, -222-3p, -221-5p, -21a-3p Expression Levels and TSHR Methylation: Diagnostic Potential and Association with Clinical and Pathological Features in Papillary Thyroid Cancer
Abstract
:1. Introduction
2. Results
2.1. Study Population
2.2. Expression of miRNA-146b-3p, -222-3p, -21a-3p, and -221-5p and Methylation of TSHR in Plasma Samples of Patients with PTC and HCs
2.3. Plasma miRNA Expression and TSHR Methylation Levels in Patients with PTC before and after Thyroidectomy
2.4. Association of TSHR Methylation Level and miRNA-221-5p, miRNA-21a-3p, miRNA-222-3p, and miRNA-146b-3p Expression with Clinicopathological Features of PTC
2.5. Correlation of miRNA Expression with TSHR Methylation Levels
2.6. The Value of miRNA-146b-3p, -222-3p, -21a-3p, -221-5p, and TSHR Methylation Assays in PTC Plasma Samples for Diagnosis of PTC
3. Discussion
4. Materials and Methods
4.1. Study Group
4.2. DNA Samples
4.3. DNA and RNA Extraction
4.4. Bisulfite Conversion and Quantitative Methylation-Specific PCR
4.5. Quantitative Reverse Transcription–Polymerase Chain Reaction
4.6. Statistical Analysis
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Characteristic | PTC n = 46 | HC n = 57 | p-Value |
---|---|---|---|
Gender | |||
Male | 8 (17.4%) | 12 (21.1%) | |
Female | 38 (82.6%) | 45 (78.9%) | |
Age at initial surgery (years) | 48.6 (14.6) | 45.21 (12.9) | p = 0.236 |
T (TNM), n (%) | - | - | |
pT1a | 15 (32.6) | ||
pT1b | 4 (8.7) | ||
pT2 | 3 (6.5) | ||
pT3a | 15 (32.6) | ||
pT3b | 9 (19.6) | ||
Tumor size (cm) | - | - | |
≤2 | 32 (69.6) | ||
>2 | 14 (30.4) | ||
Lymph node metastases at initial surgery | - | - | |
Yes | 12 (26.1) | ||
No | 34 (73.9) | ||
Variant of PTC, n (%) | - | - | |
Classical variant | 18 (39.1) | ||
Follicular variant | 10 (21.7) | ||
Diffuse sclerosing variant | 15 (32.6) | ||
Tall cell carcinoma | 3 (6.5) | ||
Extrathyroidal extension | - | - | |
Yes | 24 (52.2) | ||
No | 22 (47.8) | ||
Lymphovascular invasion | - | - | |
Yes | 25 (54.3) | ||
No | 21 (45.7) | ||
Multifocality | - | - | |
Yes | 13 (28.3) | ||
No | 33 (71.7) |
miRNA | Relative Expression 2−ΔΔCt: Median (IQR) | p | |
---|---|---|---|
Healthy Controls (n = 57) | Preoperative PTC (n = 46) | ||
146b-3p | 0.011 (0.009) | 0.016 ( 0.027) | 0.009 |
222-3p | 0.018 (0.026) | 0.038 (0.04) | <0.001 |
21a-3p | 0.072 (0.063) | 0.13 (0.21) | <0.001 |
221-5p | 0.007 (0.008) | 0.015 (0.013) | 0.003 |
miRNA | Relative Expression 2−ΔΔCt: Median (IQR) TSHR Methylation Levels: Median (IQR) | p | |
---|---|---|---|
Preoperative PTC (n = 46) | Postoperative PTC (n = 46) | ||
146b-3p | 0.016 (0.027) | 0.012 (0.011) | 0.009 |
222-3p | 0.038 (0.04) | 0.022 (0.016) | <0.001 |
21a-3p | 0.13 (0.21) | 0.069 (0.08) | 0.003 |
221-5p | 0.015 (0.013) | 0.009 (0.007) | <0.001 |
TSHR methylation | 26.041 (18.740) | 21.382 (16.220) | <0.001 |
PTC Clinicopathological Feature | Relative Expression 2−ΔΔCt: Median (IQR) | DNA Methylation Level: Median (IQR) | |||
---|---|---|---|---|---|
miRNA-146b-3p | miRNA-222-3p | miRNA-21a-3p | miRNA-221-5p | TSHR | |
Age | |||||
<55 years (n = 30; 68.2%) | 0.017 (0.026) | 0.038 (0.045) | 0.13 (0.17) | 0.014 (0.013) | 30.41(18.78) |
≥55 years (n = 16; 33.8%) | 0.018 (0.034) | 0.038 (0.021) | 0.10 (0.24) | 0.016 (0.015) | 24.09 (10.99) |
p | 0.908 | 0.963 | 0.729 | 0.344 | 0.219 |
Gender | |||||
Male (n = 8; 16.3%) | 0.021 (0.036) | 0.035 (0.033) | 0.079 (0.256) | 0.017 (0.015) | 24.09 (12.01) |
Female (n = 38; 83.7%) | 0.017 (0.246) | 0.04 (0.041) | 0.134 (0.184) | 0.013 (0.014) | 30.88 (21.32) |
p | 0.701 | 0.831 | 0.467 | 0.201 | 0.038 * |
Multifocality | |||||
Single (n = 13; 28.3%) | 0.015 (0.018) | 0.032 (0.035) | 0.103 (0.152) | 0.013 (0.013) | 30.39 (20.13) |
Multiple (≥2) (n = 33; 71.7%) | 0.033 (0.051) | 0.061 (0.046) | 0.232 (0.383) | 0.018 (0.013) | 25.04 (13.01) |
p | 0.154 | 0.016 * | 0.147 | 0.329 | 0.259 |
Extrathyroidal extension | |||||
Yes (n = 24; 71.4%) | 0.025 (0.027) | 0.028 (0.04) | 0.079 (0.112) | 0.013 (0.009) | 26.04 (11.80) |
No (n = 22; 28.6%) | 0.013 (0.02) | 0.045 (0.04) | 0.199 (0.169) | 0.0162 (0.013) | 27.07 (23.00) |
p | 0.09 | 0.132 | 0.008 * | 0.132 | 0.856 |
Lymphovascular invasion | |||||
Yes (n = 24; 52.2%) | 0.011± 0.017 | 0.031 (0.04) | 0.085 (0.119) | 0.012 (0.013) | 30.41 (19.40) |
No (n = 22; 48.8%) | 0.03 (0.028) | 0.049 (0.042) | 0.207 (0.131) | 0.016 (0.013) | 23.99 (14.78) |
p | 0.005 * | 0.087 | 0.007 * | 0.147 | 0.039 * |
T (TNM) | |||||
T1a, T1b (n = 19; 20.6%) | 0.018 ( 0.027) | 0.046 (0.042) | 0.192 (0.21) | 0.016 (0.013) | 29.34 (23.00) |
T2, T3 (n = 27; 79.4%) | 0.016 (0.26) | 0.034 (0.035) | 0.088 (0.1840 | 0.014 (0.011) | 26.04 (11.80) |
p | 0.289 | 0.220 | 0.051 | 0.326 | 0.816 |
Tumor size (cm) | |||||
≤2 (n = 32; 69.6%) | 0.017 (0.029) | 0.038 (0.046) | 0.134 (0.164) | 0.014 (0.013) | 24.23 (14.18) |
>2 (n = 14; 30.6%) | 0.018 (0.019) | 0.036 (0.030) | 0.087 (0.12) | 0.015 (0.013) | 32.64 (27.58) |
p | 0.402 | 0.272 | 0.733 | 0.412 | 0.002 * |
Lymph node metastases (preoperative PTC plasma samples) | |||||
Yes (n = 12; 25%) | 0.026 (0.056) | 0.034 ( 0.039) | 0.136 (0.372) | 0.013 (0.016) | 26.04 (17.86) |
No (n = 34; 75%) | 0.015 (0.027) | 0.04 ( 0.056) | 0.14 (0.208) | 0.013 (0.014) | 27.07 (19.92) |
p | 0.317 | 0.591 | 0.764 | 0.548 | 0.490 |
miRNA | AUC | Asymptotic 95% CI | p | miRNA’s Expression 2−ΔΔCt, TSHR Methylation Level Cut-Offs | Sensitivity | Specificity | |
---|---|---|---|---|---|---|---|
Lower Bound | Upper Bound | ||||||
146b-3p | 0.651 | 0.541 | 0.761 | 0.007 * | 0.0126 | 65.2% | 33.33% |
222-3p | 0.708 | 0.609 | 0.807 | <0.0001 * | 0.0143 | 87.9% | 52.6% |
21a-3p | 0.673 | 0.566 | 0.779 | 0.001 * | 0.1300 | 52.2% | 17.5% |
221-5p | 0.765 | 0.674 | 0.855 | <0.0001 * | 0.0103 | 66.0% | 71.7% |
TSHR methylation levels | 0.719 | 0.620 | 0.818 | <0.0001 * | 16.4960 | 86.7% | 63.2% |
Gene Symbol | DNA Strand | Primer ID | Primer Sequence (5′→3′) | Amplicon Size, bp |
---|---|---|---|---|
TSHR | Sense | Fwd | GGTGTAGAGTTGAGAATGAGGTGATTTC | 122 |
Rev | GCCCAAATCCCTAAACAAATCG | |||
Probe | FAM-ACAACACCAACTACAACAAATCCGCCGA-BHQ1 | |||
ACTB | Antisense | Fwd | TGGTGATGGAGGAGGTTTAGTAAGT | 133 |
Rev | AACCAATAAAACCTACTCCTCCCTTAA | |||
Probe | FAM-ACCACCACCCAACACACAATAACAAACACA-BHQ- 1 |
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Kazlauskiene, M.; Klimaite, R.; Kondrotiene, A.; Dauksa, A.; Dauksiene, D.; Verkauskiene, R.; Zilaitiene, B. Plasma miRNA-146b-3p, -222-3p, -221-5p, -21a-3p Expression Levels and TSHR Methylation: Diagnostic Potential and Association with Clinical and Pathological Features in Papillary Thyroid Cancer. Int. J. Mol. Sci. 2024, 25, 8412. https://doi.org/10.3390/ijms25158412
Kazlauskiene M, Klimaite R, Kondrotiene A, Dauksa A, Dauksiene D, Verkauskiene R, Zilaitiene B. Plasma miRNA-146b-3p, -222-3p, -221-5p, -21a-3p Expression Levels and TSHR Methylation: Diagnostic Potential and Association with Clinical and Pathological Features in Papillary Thyroid Cancer. International Journal of Molecular Sciences. 2024; 25(15):8412. https://doi.org/10.3390/ijms25158412
Chicago/Turabian StyleKazlauskiene, Mintaute, Raimonda Klimaite, Aiste Kondrotiene, Albertas Dauksa, Dalia Dauksiene, Rasa Verkauskiene, and Birute Zilaitiene. 2024. "Plasma miRNA-146b-3p, -222-3p, -221-5p, -21a-3p Expression Levels and TSHR Methylation: Diagnostic Potential and Association with Clinical and Pathological Features in Papillary Thyroid Cancer" International Journal of Molecular Sciences 25, no. 15: 8412. https://doi.org/10.3390/ijms25158412