Background
Methods
Clinical tissue specimens
RNA Extraction and quality control
MicroRNA microarray
Reverse transcriptase quantitative PCR
Statistical analysis
In silico analyses
Results
miRNA profiling asserts the molecular heterogeneity of OSCC
MicroRNA category | Annotated in miRBase v.16 & in v.21 (n = 41) | Proprietary to Exiqon (n = 7) |
---|---|---|
Down regulation (n = 30) (excluding invalid entries, n = 28) | miR-199a/b-3p, miR-15b, miR-16, let-7a, let-7d, miR-126, miR-203, miR-199a-5p, let-7f, miR-17, miR-106a, miR-27a, miR-130a, miR-24, miR-223, miR-143, miR-342-3p, miR-451, let-7g, miR-200a, miR-26a, let-7i, miR-15a, miR-195, miR-1259, miR-1308
| miRPlus-E1016 |
miRPlus-E1060 | ||
miRPlus-E1088 | ||
miRPlus-E1047 | ||
Up regulation (n = 18) | miR-21, miR-30e, miR-31, miR-31*, miR-193a-3p, miR-552, miR-19a, miR-19b, miR-141, miR-101, miR-144, miR-29c, miR-29b, miR-1275, miR-142-3p | miRPlus-E1245 |
miRPlus-E1194 | ||
miRPlus-F1202 |
Annotation | Moderate | Well | Difference | Fold change | p-value | ||
---|---|---|---|---|---|---|---|
Average | St.dev | Average | St.dev | ||||
hsa-miR-223 | −2.08 | 1.24 | −0.48 | 1.54 | 1.59 | 3.02 | <0.006 |
hsa-let-7f | −2.27 | 1.31 | −0.84 | 1.54 | 1.43 | 2.69 | <0.014 |
hsa-let-7d | −2.54 | 1.23 | −1.35 | 1.60 | 1.19 | 2.28 | <0.038 |
RT-qPCR validation of selected differentially expressed miRNAs
microRNA | Expression level in control samples | Expression level in OSCC samples |
P-Value |
---|---|---|---|
hsa-let-7a | 1.00 (0.94–1.45) a
| 0.46 (0.37–0.58) a
|
<0.0001
|
hsa-let-7d | 1.00 (0.73–1.42) a
| 0.44 (0.36–0.58) a
|
<0.0001
|
hsa-let-7f | 1.00 (0.86–1.26) a
| 0.49 (0.41–0.65) a
|
<0.0001
|
hsa-miR-16 | 1.00 (0.93–1.09) a
| 0.66 (0.44–0.92) a
|
0.008
|
hsa-miR-29b | 1.00 (0.87–1.08) a
| 1.58 (1.02–1.97) a
|
0.026
|
hsa-miR-142-3p | 1.00 (0.56–1.67) a
| 5.94 (3.26–10.20) a
|
<0.0001
|
hsa-miR-144 | 1.67 (0.67–2.67) b
| 14.26 (3.19–25.34) b
|
0.03
|
hsa-miR-203 | 1.00 (0.64–1.58) a
| 1.82 (0.99–3.57) a
|
0.03
|
hsa-miR-223 | 0.97 (0.56–1.38) b
| 2.67 (2.08–3.26) b
|
<0.0001
|
hsa-miR-1275 | 1.05 (0.84–1.25) b
| 1.33 (1.09–1.57) b
| 0.06 |
Association of miRNA expression levels with tumour characteristics
Variable | let-7a | let-7d | let-7f | miR-16 | miR-29b | miR-142-3p | miR-144 | miR-223 | miR-203 | miR-1275 |
---|---|---|---|---|---|---|---|---|---|---|
Age (in yrs.) | ||||||||||
<60 | 0.49 ± 0.22 | 0.44 (0.11–1.25) | 0.51 (0.2–1.25) | 0.6 (0.01–1.45) | 1.58 (0.02–7.79) | 5.76 (0.07–39.44) | 10.05 ± 14.29 | 2.54 ± 2.12 | 1.95 (0–9.25) | 1.37 ± 0.99 |
≥60 | 0.49 ± 0.17 | 0.43 (0.26–2.98) | 0.49 (0.31–2.20) | 0.68 (0.22–2.48) | 1.59 (0.21–5.37) | 6.24 (0.29–17.81) | 19.94 ± 64.44 | 2.86 ± 2.56 | 1.72 (0.13–11.01) | 1.28 ± 0.84 |
P value
| 0.92 | 0.69 | 0.92 | 0.74 | 0.63 | 0.33 | 0.45 | 0.6 | 0.32 | 0.72 |
Gender | ||||||||||
Male | 0.54 ± 0.16 | 0.51 (0.31–2.98) | 0.55 (0.22–2.2) | 0.68 (0.22–2.48) | 1.42 (0.02–7.79) | 5.61 (0.07–13.41) | 6.79 ± 7.35 | 2.54 ± 2.19 | 1.89 (0.13–9.25) | 1.15 ± 0.55 |
Female | 0.45 ± 0.21 | 0.42 (0.11–1.63) | 0.45 (0.2–1.25) | 0.55 (0.01–1.45) | 1.59 (0.25–7.18) | 6.51 (0.69–39.44) | 19.82 ± 56.45 | 2.78 ± 2.41 | 1.73 (0–11.01) | 1.47 ± 1.11 |
P value
| 0.06 | 0.028 | 0.08 | 0.58 | 0.35 | 0.07 | 0.19 | 0.69 | 0.41 | 0.15 |
Anatomical site | ||||||||||
GBC | 0.47 ± 0.20 | 0.42 (0.11–2.98) | 0.46 (0.2–2.2) | 0.65 (0.01–2.48) | 1.56 (0.02–7.79) | 6.33 (0.07–39.44) | 16.03 ± 47.12 | 2.74 ± 2.16 | 1.72 (0–11.01) | 1.34 ± 0.98 |
Tongue | 0.60 ± 0.14 | 0.52 (0.35–0.76) | 0.59 (0.47–0.92) | 0.66 (0.42–1.32) | 1.63 (0.54–2.44) | 4.36 (0.48–9.37) | 5.28 ± 5.06 | 2.31 ± 3.05 | 2.69 (0.36–4.3) | 1.28 ± 0.61 |
P value
| 0.05 | 0.16 | 0.01 | 0.85 | 0.99 | 0.09 | 0.12 | 0.59 | 0.25 | 0.84 |
Clinical stage | ||||||||||
I or II | 0.60 ± 0.20 | 0.55 (0.34–0.82) | 0.67 (0.43–0.78) | 0.77 (0.4–1.11) | 0.84 (0.58–1.61) | 2.17 (0.48–9.37) | 2.80 ± 2.39 | 1.17 ± 0.63 | 3.14 (1.73–6.14) | 0.92 ± 0.26 |
III or IV | 0.49 ± 0.20 | 0.44 (0.23–2.98) | 0.51 (0.22–2.2) | 0.6 (0.01–2.48) | 1.61 (0.02–7.79) | 5.94 (0.07–39.44) | 17.11 ± 48.91 | 2.84 ± 2.49 | 1.82 (0.26–9.25) | 1.40 ± 1.01 |
P value
| 0.33 | 0.63 | 0.39 | 0.68 | 0.06 | 0.1 | 0.05 |
0.004
| 0.17 | 0.03 |
Tumour Size | ||||||||||
T1 or T2 | 0.56 ± 0.16 | 0.44 (0.34–0.82) | 0.58 (0.42–0.78) | 0.79 (0.4–1.13) | 1.01 (0.54–1.70) | 2.93 (0.48–15.76) | 3.56 ± 3.52 | 0.95 ± 0.62 | 2.73 (0.53–7.9) | 1.02 ± 0.28 |
T3 or T4 | 0.49 ± 0.21 | 0.46 (0.23–2.98) | 0.49 (0.22–2.2) | 0.55 (0.01–2.48) | 1.67 (0.02–7.79) | 5.94 (0.07–39.44) | 19.02 ± 52.15 | 3.14 ± 2.53 | 1.75 (0.26–9.25) | 1.44 ± 1.07 |
P value
| 0.31 | 0.91 | 0.23 | 0.13 | 0.006 | 0.21 | 0.07 |
≤
0.0001
| 0.11 | 0.03 |
Nodal invasion | ||||||||||
N- | 0.60 ± 0.18 | 0.55 (0.34–0.82) | 0.67 (0.43–0.98) | 0.64 (0.4–1.11) | 0.84 (0.45–1.61) | 1.84 (0.48–9.37) | 2.20 ± 2.09 | 1.48 ± 0.85 | 4.24 (1.73–9.25) | 0.85 ± 0.23 |
N+ | 0.49 ± 0.20 | 0.44 (0.23–2–98) | 0.51 (0.22–2.2) | 0.6 (0.01–2.48) | 1.65 (0.02–7.79) | 6.15 (0.07–39.44) | 17.82 ± 49.88 | 2.87 ± 2.54 | 1.75 (0.26–7.9) | 1.43 ± 1.02 |
P value
| 0.22 | 0.63 | 0.31 | 0.78 | 0.009 | 0.007 | 0.04 | 0.01 | 0.01 |
0.003
|
Histological grade | ||||||||||
G1/well | 0.53 ± 0.25 | 0.43 (0.23–1.63) | 0.46 (0.22–1.25) | 0.67 (0.22–1.45) | 1.59 (0.02–7.79) | 7.52 (0.07–39.44) | 21.97 ± 68.38 | 3.42 ± 2.95 | 2.21 (0.26–11.01) | 1.43 ± 0.88 |
G2/moderate | 0.47 ± 0.13 | 0.44 (0.26–0.85) | 0.52 (0.34–0.98) | 0.57 (0.01–1.13) | 1.60 (0.45–7.18) | 5.28 (0.67–15.76) | 9.79 ± 14.40 | 1.99 ± 1.33 | 1.74 (0.28–9.25) | 1.18 ± 0.97 |
P value
| 0.34 | 0.78 | 0.96 | 0.23 | 0.77 | 0.25 | 0.41 | 0.04 | 0.42 | 0.33 |
Smoking | ||||||||||
Yes | 0.56 ± 18 | 0.46 (0.32–2.98) | 0.55 (0.22–2.2) | 0.68 (0.22–2.48) | 1.23 (0.02–4.92) | 3.6 (0.07–13.41) | 4.72 ± 4.76 | 2.62 ± 2.54 | 1.96 (0.27–9.25) | 1.06 ± 0.59 |
No | 0.48 ± 0.20 | 0.44 (0.23–1.63) | 0.47 (0.27–1.25) | 0.59 (0.01–1.45) | 1.64 (0.25–7.79) | 6.27 (0.69–39.44) | 18.90 ± 52.86 | 2.73 ± 2.38 | 1.84 (0.26–7.90) | 1.43 ± 1.05 |
P value
| 0.21 | 0.43 | 0.12 | 0.62 | 0.07 | 0.03 | 0.1 | 0.88 | 0.72 | 0.11 |
Tobacco chewing | ||||||||||
Yes | 0.47 ± 0.17 | 0.44 (0.23–2.98) | 0.46 (0.27–2.2) | 0.65 (0.01–2.48) | 1.61 (0.25–7.18) | 5.94 (0.69–17.81) | 19.41 ± 54.98 | 2.89 ± 2.65 | 1.72 (0.26–7.9) | 1.46 ± 1.07 |
No | 0.55 ± 0.25 | 0.43 (0.26–1.25) | 0.55 (0.22–1.25) | 0.66 (0.22–1.45) | 1.25 (0.02–7.79) | 4.32 (0.07–39.44) | 6.10 ± 5.51 | 2.31 ± 1.76 | 3.44 (0.36–9.25) | 1.04 ± 0.62 |
P value
| 0.22 | 0.74 | 0.24 | 0.66 | 0.27 | 0.13 | 0.15 | 0.42 | 0.06 | 0.07 |
Alcohol intake | ||||||||||
Yes | 0.52 ± 0.16 | 0.57 (0.32–2.98) | 0.53 (0.22–2.2) | 0.66 (0.22–2.48) | 1.65 (0.02–4.92) | 5.46 (0.07–13.41) | 7.15 ± 7.41 | 2.97 ± 2.59 | 1.75 (0.27–5.97) | 1.17 ± 0.61 |
No | 0.49 ± 0.21 | 0.42 (0.23–1.63) | 0.47 (0.27–1.25) | 0.65 (0.01–1.45) | 1.56 (0.25–7.79) | 6.38 (0.48–39.44) | 18.32 ± 53.62 | 2.60 ± 2.35 | 2.09 (0.26–9.25) | 1.39 ± 1.07 |
P value
| 0.55 | 0.06 | 0.38 | 0.92 | 0.94 | 0.3 | 0.21 | 0.62 | 0.54 | 0.34 |