Background
Methods
Study cohort and tissue sampling
Patient information | Lymph node negative cases (16) | Lymph node positive cases (19) |
---|---|---|
Age (years)
| ||
Average (±SD) | 58 ± 14 | 54 ± 14 |
Range | 36–84 | 28–78 |
<50 | 4 (25%) | 9 (47.3%) |
50-69 | 8 (50%) | 7 (36.8%) |
>69 | 4 (25%) | 3 (15.8%) |
Tumour size (mm)
| ||
Average (±SD) | 32 ± 12 | 32 ± 21 |
Range | 12–60 | 12–100 |
<20 | 2 (12.5%) | 4 (21%) |
20-39 | 10 (62.5%) | 12 (63.2%) |
>39 | 4 (25%) | 3 (15.8%) |
No. of positive lymph nodes
| ||
1-3 | 0 | 13 (68.4%) |
-Micrometastases (<2 mm) (No LN available) | 0 | 4/13 (30.7%) |
>3 | 0 | 6 (31.6%) |
Normal adjacent tissue
| ||
Yes | 9 (56.3%) | 15 (78.9%) |
No | 7 (43.8%) | 4 (21.1%) |
Extraction of RNA
miRNA arrays
miRNA array analysis
Semi-quantitative real-time PCR
Statistical analysis
Results
miRNAs differentially expressed in TNBC
Systematic name | Fold regulation | p-value |
---|---|---|
hsa-miR-130a | −2.21 | 0.0160 |
hsa-miR-1280 | 2.10 | 3.11E-04 |
hsa-miR-590-5p | 2.15 | 0.0131 |
hsa-miR-1308 | 2.28 | 0.0035 |
hsa-miR-17* | 3.03 | 3.11E-04 |
miRNAs differentially expressed in LN negative IDCs compared to LN positive IDCs
miRNAs associated with LN metastasis
Lymph node positive cases | Lymph node negative cases | |||||
---|---|---|---|---|---|---|
IDC v NAT | LN met v NAT | IDC v NAT | ||||
Systematic name | Fold change | p-value | Fold change | p-value | Fold change | p-value |
hsa-let-7a | −2.36 | 0.00419 | −3.33 | 0.00042 | 1.05 | ns |
hsa-let-7b | −2.99 | 0.00011 | −4.67 | 0.00007 | −1.30 | ns |
hsa-let-7c | −3.84 | 0.00006 | −6.34 | 0.00001 | −1.72 | ns |
hsa-miR-100 | −4.37 | 0.00176 | −4.57 | 0.00331 | −1.84 | ns |
hsa-miR-101 | −2.61 | 0.01750 | −3.30 | 0.02278 | 1.22 | ns |
hsa-miR-10a | −2.37 | 0.02228 | −4.64 | 0.00496 | −1.78 | ns |
hsa-miR-125b | −5.18 | 0.00010 | −8.41 | 0.00001 | −2.69 | ns |
hsa-miR-126 | −2.72 | 0.00451 | −3.76 | 0.00451 | −1.50 | ns |
hsa-miR-126* | −2.31 | 0.04409 | −2.63 | 0.02725 | −1.56 | ns |
hsa-miR-130a | −3.32 | 0.01446 | −6.61 | 0.00430 | −1.32 | ns |
hsa-miR-135b | 14.14 | 0.00012 | 6.92 | 0.02725 | 14.87 | ns |
hsa-miR-136 | −6.18 | 0.00011 | −9.58 | 0.00223 | −3.42 | ns |
hsa-miR-143 | −2.99 | 0.01750 | −3.04 | 0.03718 | −2.02 | ns |
hsa-miR-195 | −5.70 | 0.00006 | −10.02 | 0.00029 | −2.50 | ns |
hsa-miR-1977_v14.0 | −2.28 | 0.00377 | −2.91 | 0.00069 | −1.49 | ns |
hsa-miR-199a-3p | −2.50 | 0.01750 | −4.84 | 0.00451 | −1.55 | ns |
hsa-miR-199a-5p | −2.46 | 0.04409 | −4.64 | 0.00869 | −1.66 | ns |
hsa-miR-199b-5p | −5.66 | 0.00011 | −9.29 | 0.00021 | −3.40 | ns |
hsa-miR-205 | −4.65 | 0.02890 | −5.18 | 0.01648 | −1.82 | ns |
hsa-miR-210 | 4.56 | 0.00862 | 2.98 | 0.02389 | 4.01 | ns |
hsa-miR-214 | −2.64 | 0.01360 | −3.64 | 0.02023 | −2.28 | ns |
hsa-miR-26a | −2.67 | 0.01428 | −3.84 | 0.00247 | −1.00 | ns |
hsa-miR-26b | −2.43 | 0.01681 | −3.01 | 0.01308 | −1.01 | ns |
hsa-miR-29c | −2.33 | 0.04343 | −2.69 | 0.03676 | −1.12 | ns |
hsa-miR-320c | −2.15 | 0.02461 | −2.65 | 0.02023 | −1.02 | ns |
hsa-miR-34a | −2.12 | 0.02087 | −3.59 | 0.00825 | −1.11 | ns |
hsa-miR-497 | −5.11 | 0.00006 | −7.91 | 0.00029 | −2.06 | ns |
Biological functions and pathways regulated by the 27 miRNAs associated with LN metastasis
Pathway | Homo sapiens reference genome (No. of genes) | miRNA target genes (No. of genes) | Expected | Over/under represented (+/−) | p value |
---|---|---|---|---|---|
Wnt signaling pathway | 318 | 16 | 5.69 | + | 0.000248 |
TGF-beta signaling pathway | 149 | 10 | 2.66 | + | 0.000423 |
Cell cycle | 22 | 4 | 0.39 | + | 0.00072 |
p53 pathway | 114 | 7 | 2.04 | + | 0.00487 |
p53 pathway by glucose deprivation | 25 | 3 | 0.45 | + | 0.0106 |
p53 pathway feedback loops | 52 | 4 | 0.93 | + | 0.0148 |
PI3 kinase pathway | 117 | 6 | 2.09 | + | 0.0198 |
Heterotrimeric G-protein signaling pathway-Gi alpha and Gs alpha mediated pathway | 164 | 7 | 2.93 | + | 0.0296 |
EGF receptor signaling pathway | 130 | 6 | 2.32 | + | 0.0308 |
De novo pyrmidine ribonucleotides biosythesis | 18 | 2 | 0.32 | + | 0.0418 |
Correlation between miRNA expression and clinical parameters
Age at diagnosis | Tumour size (mm) | % + ve lymph nodes | ||||
---|---|---|---|---|---|---|
Spearman rank | p-value | Spearman rank | p-value | Spearman rank | p-value | |
let-7a
| 0.04597 | 0.7932 | 0.07885 | 0.6525 | −0.2904 | 0.0906 |
let-7b
| −0.08997 | 0.6073 | 0.08432 | 0.6301 | −0.3324 | 0.0511 |
miR-100
| −0.09418 | 0.5905 | 0.171 | 0.3259 | −0.1892 | 0.2764 |
miR-101
| 0.1756 | 0.313 | 0.17 | 0.3288 |
−0.4355
|
0.0089
|
miR-126*
| 0.0471 | 0.7882 | 0.08945 | 0.6093 | −0.1581 | 0.3643 |
miR-130a
| 0.07147 | 0.6833 | −0.1973 | 0.256 | −0.2539 | 0.1411 |
miR-210
| 0.1029 | 0.5565 | 0.004349 | 0.9802 | −0.09827 | 0.5744 |
miR-26a
| 0.05199 | 0.7668 | 0.08586 | 0.6238 | −0.1696 | 0.3301 |
miR-26b
| 0.1511 | 0.3863 | 0.05724 | 0.744 | −0.1778 | 0.3068 |
miR-29c
| 0.07231 | 0.6797 | 0.1254 | 0.4728 | −0.3131 | 0.0671 |