Background
Methods
Patient samples
RNA isolation
Gene expression microarray assay analysis
MiRNA microarray assay analysis
Reverse transcription and quantitative real-time PCR
Overrepresentation analysis
Statistical analysis
Results
Patient characteristics
Correlation analysis of miRNA and mRNA between MFS patients and HV controls
Differentially expressed miRNAs between MFS patients and HV controls
miRNA | Median log2 MFS | Median log2 HVS | Fold change | Log2 fold change | Regulation | P value | Adjusted P value | AUC |
---|---|---|---|---|---|---|---|---|
hsa-miR-4271 | − 3.32 | 4.40 | 0.0047 | − 7.72 | Down | 0.0037 | 0.0689 | 0.95 |
hsa-miR-3616-3p | − 3.32 | 4.15 | 0.0056 | − 7.47 | Down | 0.0003 | 0.0149 | 0.96 |
hsa-miR-1228 | − 3.32 | 3.80 | 0.0072 | − 7.12 | Down | 0.0004 | 0.0149 | 0.98 |
hsa-miR-1238 | − 3.32 | 3.17 | 0.0111 | − 6.49 | Down | 0.0169 | 0.1552 | 0.84 |
hsa-miR-191 | − 3.32 | 3.03 | 0.0123 | − 6.35 | Down | 0.0450 | 0.2177 | 0.80 |
hsa-miR-1234 | 0.94 | 4.02 | 0.12 | − 3.09 | Down | 0.0004 | 0.0149 | 0.98 |
hsa-miR-4313 | 0.69 | 3.39 | 0.15 | − 2.70 | Down | 0.0468 | 0.2177 | 0.78 |
hsa-miR-139-3p | 0.65 | 3.15 | 0.18 | − 2.50 | Down | 0.0001 | 0.0149 | 1.00 |
hsa-miR-874 | 2.83 | 4.76 | 0.26 | − 1.92 | Down | 0.0042 | 0.0731 | 0.92 |
hsa-miR-564 | 4.70 | 5.90 | 0.43 | − 1.20 | Down | 0.0003 | 0.0149 | 1.00 |
hsa-miR-940 | 3.06 | 4.21 | 0.45 | − 1.15 | Down | 0.0004 | 0.0149 | 1.00 |
hsa-miR-1207-5p | 5.80 | 6.93 | 0.46 | − 1.13 | Down | 0.0319 | 0.1878 | 0.83 |
hsa-miR-1280 | 1.64 | 2.73 | 0.47 | − 1.10 | Down | 0.0048 | 0.0790 | 0.98 |
hsa-miR-181d | 3.93 | 4.99 | 0.48 | − 1.06 | Down | 0.0277 | 0.1845 | 0.80 |
hsa-miR-1275 | 3.77 | 4.80 | 0.49 | − 1.03 | Down | 0.0014 | 0.0323 | 0.94 |
hsa-miR-3653 | 7.04 | 8.01 | 0.51 | − 0.97 | Down | 0.0484 | 0.2177 | 0.85 |
hsa-miR-1268 | 5.18 | 6.07 | 0.54 | − 0.88 | Down | 0.0239 | 0.1739 | 0.89 |
hsa-miR-320b | 10.66 | 11.48 | 0.57 | − 0.82 | Down | 0.0085 | 0.1242 | 0.92 |
hsa-miR-532-3p | 8.69 | 9.46 | 0.59 | − 0.77 | Down | 0.0130 | 0.1444 | 0.84 |
hsa-miR-642b | 3.17 | 3.93 | 0.59 | − 0.76 | Down | 0.0179 | 0.1552 | 0.87 |
hsa-miR-4323 | 2.73 | 3.44 | 0.61 | − 0.70 | Down | 0.0218 | 0.1675 | 0.87 |
hsa-miR-93 | 5.67 | 6.31 | 0.64 | − 0.64 | Down | 0.0297 | 0.1871 | 0.80 |
hsa-miR-3162 | 6.83 | 7.46 | 0.65 | − 0.63 | Down | 0.0068 | 0.1050 | 0.89 |
hsa-miR-3679-5p | 4.53 | 5.13 | 0.66 | − 0.59 | Down | 0.0278 | 0.1845 | 0.87 |
hsa-miR-423-5p | 9.11 | 9.62 | 0.70 | − 0.51 | Down | 0.0124 | 0.1433 | 0.86 |
hsa-miR-1225-5p | 6.16 | 6.67 | 0.70 | − 0.51 | Down | 0.0287 | 0.1852 | 0.84 |
hsa-miR-3651 | 4.99 | 5.44 | 0.73 | − 0.45 | Down | 0.0478 | 0.2177 | 0.81 |
hsa-miR-638 | 5.98 | 6.41 | 0.74 | − 0.43 | Down | 0.0193 | 0.1575 | 0.86 |
hsa-miR-766 | 4.84 | 5.25 | 0.76 | − 0.40 | Down | 0.0238 | 0.1739 | 0.91 |
hsa-miR-191 | 5.10 | 5.49 | 0.76 | − 0.39 | Down | 0.0350 | 0.1978 | 0.83 |
hsa-miR-762 | 4.55 | 4.73 | 0.88 | − 0.18 | Down | 0.0403 | 0.2106 | 0.78 |
hsa-miR-221 | 2.77 | − 3.32 | 68.19 | 6.09 | Up | 0.0499 | 0.2177 | 0.18 |
hsa-miR-1288 | 2.53 | − 3.32 | 57.95 | 5.86 | Up | 0.0344 | 0.1978 | 0.21 |
hsa-miR-3125 | 2.35 | − 3.32 | 50.81 | 5.67 | Up | 0.0481 | 0.2177 | 0.21 |
hsa-miR-500b | 2.27 | − 3.32 | 48.35 | 5.60 | Up | 0.0142 | 0.1462 | 0.14 |
hsa-miR-200c | 2.13 | − 3.32 | 43.66 | 5.45 | Up | 0.0142 | 0.1462 | 0.14 |
hsa-miR-3200-3p | 2.07 | − 3.32 | 41.98 | 5.39 | Up | 0.0280 | 0.1845 | 0.17 |
hsa-miR-3667-5p | 1.81 | − 3.32 | 35.05 | 5.13 | Up | 0.0380 | 0.2067 | 0.14 |
hsa-miR-627 | 1.74 | − 3.32 | 33.47 | 5.06 | Up | 0.0311 | 0.1878 | 0.14 |
hsa-miR-664 | 1.33 | − 3.32 | 25.09 | 4.65 | Up | 0.0395 | 0.2106 | 0.20 |
hsa-miR-223 | 13.58 | 11.98 | 3.04 | 1.60 | Up | 0.0496 | 0.2177 | 0.15 |
hsa-miR-660 | 4.28 | 2.92 | 2.57 | 1.36 | Up | 0.0192 | 0.1575 | 0.08 |
hsa-miR-29c | 4.59 | 3.33 | 2.41 | 1.27 | Up | 0.0177 | 0.1552 | 0.00 |
hsa-miR-7 | 4.62 | 3.56 | 2.09 | 1.06 | Up | 0.0357 | 0.1978 | 0.12 |
hsa-miR-29a | 6.93 | 5.98 | 1.93 | 0.95 | Up | 0.0117 | 0.1407 | 0.04 |
hsa-miR-500a | 5.70 | 4.80 | 1.87 | 0.90 | Up | 0.0008 | 0.0202 | 0.04 |
hsa-miR-23a | 9.46 | 8.57 | 1.85 | 0.89 | Up | 0.0103 | 0.1364 | 0.06 |
hsa-miR-151-5p | 9.11 | 8.33 | 1.73 | 0.79 | Up | 0.0175 | 0.1552 | 0.09 |
hsa-miR-324-5p | 6.20 | 5.41 | 1.72 | 0.79 | Up | 0.0005 | 0.0149 | 0.05 |
hsa-miR-4306 | 12.57 | 11.84 | 1.66 | 0.73 | Up | 0.0161 | 0.1552 | 0.08 |
hsa-miR-186 | 6.78 | 6.10 | 1.60 | 0.68 | Up | 0.0261 | 0.1845 | 0.12 |
hsa-miR-502-3p | 5.53 | 4.87 | 1.58 | 0.66 | Up | 0.0005 | 0.0149 | 0.00 |
hsa-miR-23b | 6.34 | 5.70 | 1.56 | 0.64 | Up | 0.0468 | 0.2177 | 0.18 |
hsa-miR-629 | 4.76 | 4.12 | 1.56 | 0.64 | Up | 0.0466 | 0.2177 | 0.20 |
hsa-miR-362-5p | 6.34 | 5.74 | 1.52 | 0.60 | Up | 0.0005 | 0.0149 | 0.02 |
hsa-miR-652 | 9.07 | 8.47 | 1.51 | 0.60 | Up | 0.0116 | 0.1407 | 0.16 |
hsa-miR-24 | 7.95 | 7.38 | 1.48 | 0.57 | Up | 0.0018 | 0.0392 | 0.01 |
hsa-miR-501-3p | 4.31 | 3.74 | 1.48 | 0.56 | Up | 0.0099 | 0.1364 | 0.04 |
hsa-miR-30e | 5.97 | 5.49 | 1.39 | 0.48 | Up | 0.0027 | 0.0542 | 0.08 |
hsa-miR-331-3p | 10.09 | 9.62 | 1.38 | 0.46 | Up | 0.0205 | 0.1621 | 0.16 |
hsa-miR-451 | 16.51 | 16.05 | 1.38 | 0.46 | Up | 0.0004 | 0.0149 | 0.06 |
hsa-miR-532-5p | 5.94 | 5.53 | 1.33 | 0.41 | Up | 0.0318 | 0.1878 | 0.13 |
hsa-miR-103 | 10.66 | 10.25 | 1.33 | 0.41 | Up | 0.0478 | 0.2177 | 0.18 |
Differentially expressed genes between MFS patients and HV controls
Gene Name | NCBI accession code | Median log2 MFS | Median log2 HVS | Fold change | log2 Fold change | Regulation | P value | AUC |
---|---|---|---|---|---|---|---|---|
POT1 (protection of telomeres 1) | NM_015450 | 7.96 | 9.05 | 0.47 | −1.09 | Down | 0.0002 | 1.00 |
SIRPB1 (signal regulatory protein beta 1) | NM_001135844 | 11.30 | 8.96 | 5.08 | 2.34 | Up | 0.0474 | 0.16 |
HBZ (hemoglobin subunit zeta) | NM_005332 | 8.71 | 6.47 | 4.73 | 2.24 | Up | 0.0132 | 0.16 |
MYOM2 (myomesin 2) | NM_003970 | 8.38 | 6.50 | 3.67 | 1.87 | Up | 0.0372 | 0.14 |
ITGB3 (integrin subunit beta 3) | NM_000212 | 9.23 | 7.38 | 3.62 | 1.86 | Up | 0.0392 | 0.08 |
MX1 (MX dynamin like GTPase 1) | NM_002462 | 12.89 | 11.36 | 2.89 | 1.53 | Up | 0.0383 | 0.16 |
DYSF (dysferlin) | NM_003494 | 10.49 | 9.04 | 2.74 | 1.45 | Up | 0.0149 | 0.08 |
CCR1 (C-C motif chemokine receptor 1) | NM_001295 | 12.91 | 11.50 | 2.66 | 1.41 | Up | 0.0217 | 0.12 |
IFIT2 (interferon induced protein with tetratricopeptide repeats 2) | NM_001547 | 11.56 | 11.33 | 1.17 | 0.23 | Up | 0.0317 | 0.14 |
LRG1 (leucine rich alpha-2-glycoprotein 1) | NM_052972 | 11.14 | 9.85 | 2.45 | 1.29 | Up | 0.0181 | 0.16 |
TRANK1 (tetratricopeptide repeat and ankyrin repeat containing 1) | NM_014831 | 10.86 | 9.62 | 2.37 | 1.25 | Up | 0.0013 | 0.02 |
ZAN (zonadhesin (gene/pseudogene)) | NM_173059 | 9.30 | 8.08 | 2.34 | 1.23 | Up | 0.0423 | 0.18 |
CRYAA (crystallin alpha A) | NM_000394 | 9.30 | 8.08 | 2.34 | 1.23 | Up | 0.0329 | 0.18 |
NRGN (neurogranin) | NM_006176 | 12.69 | 11.52 | 2.25 | 1.17 | Up | 0.0356 | 0.12 |
RNF213 (ring finger protein 213) | NM_020914 | 8.24 | 7.70 | 1.46 | 0.55 | Up | 0.0377 | 0.18 |
LIMK2 (LIM domain kinase 2) | NM_001031801 | 9.31 | 8.16 | 2.22 | 1.15 | Up | 0.0181 | 0.14 |
MFN2 (mitofusin 2) | NM_014874 | 5.49 | 5.18 | 1.24 | 0.31 | Up | 0.0288 | 0.20 |
CTTN (cortactin) | NM_005231 | 9.51 | 8.36 | 2.21 | 1.14 | Up | 0.0268 | 0.16 |
MX2 (MX dynamin like GTPase 2) | NM_002463 | 12.60 | 11.52 | 2.11 | 1.07 | Up | 0.0157 | 0.12 |
CD14 (CD14 molecule) | NM_001174104 | 15.04 | 13.97 | 2.10 | 1.07 | Up | 0.0332 | 0.16 |
GRN (granulin precursor) | NM_002087 | 15.83 | 14.76 | 2.10 | 1.07 | Up | 0.0258 | 0.16 |
RNF222 (ring finger protein 222) | NM_001146684 | 5.29 | 5.01 | 1.22 | 0.28 | Up | 0.0336 | 0.14 |
MVP (major vault protein) | NM_017458 | 14.55 | 13.49 | 2.08 | 1.06 | Up | 0.0046 | 0.08 |
CXCL5 (C-X-C motif chemokine ligand 5) | NM_002994 | 8.88 | 7.83 | 2.07 | 1.05 | Up | 0.0455 | 0.18 |
CTNNA1 (catenin alpha 1) | NM_001903 | 9.08 | 8.05 | 2.05 | 1.04 | Up | 0.0309 | 0.16 |
MMP9 (matrix metallopeptidase 9) | NM_004994 | 13.20 | 12.14 | 2.07 | 1.05 | Up | 0.0456 | 0.20 |
SOCS3 (suppressor of cytokine signaling 3) | NM_003955 | 9.47 | 8.43 | 2.05 | 1.03 | Up | 0.0472 | 0.20 |
GBP2 (guanylate binding protein 2) | NM_004120 | 11.49 | 10.46 | 2.04 | 1.03 | Up | 0.0097 | 0.08 |
GNL3L (G protein nucleolar 3 like) | NM_019067 | 8.33 | 7.93 | 1.32 | 0.41 | Up | 0.0325 | 0.20 |
CLU (clusterin) | NM_001831 | 9.71 | 8.69 | 2.02 | 1.02 | Up | 0.0423 | 0.22 |
SELL (selectin L) | NM_000655 | 14.93 | 13.93 | 2.00 | 1.00 | Up | 0.0011 | 0.04 |
OSM (oncostatin M) | NM_020530 | 9.18 | 8.19 | 2.00 | 1.00 | Up | 0.0414 | 0.14 |
Validation of candidate miRNAs and mRNAs by RT-qPCR
Inverse correlation between miRNA and target mRNA
miRNA | mRNA | P value correlation | Correlation | Fold change miRNA | P value miRNA | Fold change mRNA | P value mRNA |
---|---|---|---|---|---|---|---|
hsa-miR-1234 | LIMK2 | 0.00001 | − 0.89 | 0.12 | 0.00038 | 2.22 | 0.01813 |
hsa-miR-1234 | DYSF | 0.00018 | − 0.85 | 0.12 | 0.00038 | 2.74 | 0.01491 |
hsa-miR-1234 | GBP2 | 0.00143 | − 0.78 | 0.12 | 0.00038 | 2.04 | 0.00973 |
hsa-miR-1234 | MMP9 | 0.00382 | − 0.74 | 0.12 | 0.00038 | 2.15 | 0.03885 |
hsa-miR-324-5p | POT1 | 0.00670 | − 0.69 | 1.72 | 0.00050 | 0.47 | 0.00017 |
hsa-miR-151-5p | POT1 | 0.00728 | − 0.68 | 1.73 | 0.01754 | 0.47 | 0.00017 |
hsa-miR-200c | POT1 | 0.01132 | − 0.65 | 43.66 | 0.01425 | 0.47 | 0.00017 |
hsa-miR-362-5p | POT1 | 0.01281 | − 0.64 | 1.52 | 0.00045 | 0.47 | 0.00017 |
hsa-miR-502-3p | POT1 | 0.01590 | − 0.63 | 1.58 | 0.00054 | 0.47 | 0.00017 |
hsa-miR-500b | POT1 | 0.01868 | − 0.62 | 48.35 | 0.01419 | 0.47 | 0.00017 |
hsa-miR-627 | POT1 | 0.01868 | − 0.62 | 33.47 | 0.03109 | 0.47 | 0.00017 |
Correlation between the clinical parameters and expression levels of miRNA and mRNA
Parameters | Marfan syndrome patients | Healthy volunteers | |||||||
---|---|---|---|---|---|---|---|---|---|
Age (MFS patients) | Aortic root status (Z score) | LVEDD | MVP | Age | |||||
miRNA | Correlation | P value | Correlation | P value | Correlation | P value | P value* | Correlation | P value |
hsa-miR-1234 | 0.003 | 0.988 | 0.063 | 0.761 | − 0.075 | 0.716 | 0.732 | − 0.122 | 0.554 |
hsa-miR-151-5p | 0.519 |
0.007
| 0.375 |
0.059
| 0.496 |
0.010
| 0.979 | 0.055 | 0.789 |
hsa-miR-200c | 0.370 |
0.063
| 0.473 |
0.015
| 0.213 | 0.295 | 0.060 | − 0.220 | 0.279 |
hsa-miR-24 | 0.496 |
0.010
| 0.281 | 0.164 | 0.420 |
0.033
| 0.654 | − 0.194 | 0.342 |
hsa-miR-30e | 0.565 |
0.003
| 0.230 | 0.258 | 0.472 |
0.015
| 0.517 | − 0.030 | 0.883 |
hsa-miR-324-5p | 0.510 |
0.008
| 0.350 |
0.079
| 0.457 |
0.019
| 0.391 | − 0.019 | 0.927 |
hsa-miR-362-5p | 0.413 |
0.036
| 0.216 | 0.289 | 0.383 |
0.053
| 0.673 | 0.024 | 0.906 |
hsa-miR-500b | 0.440 |
0.024
| 0.384 |
0.053
| 0.456 |
0.019
| 0.812 | − 0.003 | 0.987 |
hsa-miR-502-3p | 0.505 |
0.008
| 0.260 | 0.200 | 0.425 |
0.030
| 0.816 | 0.036 | 0.860 |
hsa-miR-627 | 0.310 | 0.123 | 0.291 | 0.149 | 0.423 |
0.032
| 0.816 | 0.010 | 0.961 |
hsa-miR-331-3p | 0.307 | 0.127 | 0.333 | 0.097 | 0.015 | 0.942 | 0.041 | − 0.243 | 0.231 |
Parameters | Marfan syndrome patients | Healthy volunteers | |||||||
---|---|---|---|---|---|---|---|---|---|
Age | Aortic root status (Z score) | LVEDD | MVP | Age | |||||
mRNA | Correlation | P value | Correlation | P value | Correlation | P value | P value | Correlation | P value |
DYSF | 0.123 | 0.550 | 0.087 | 0.672 | − 0.010 | 0.963 | 0.958 | 0.186 | 0.385 |
GBP2 | 0.200 | 0.326 | 0.071 | 0.729 | − 0.198 | 0.332 | 0.816 | 0.080 | 0.710 |
LIMK2 | 0.003 | 0.989 | − 0.142 | 0.489 | − 0.241 | 0.235 | 0.916 | 0.135 | 0.530 |
MMP9 | 0.276 | 0.173 | 0.272 | 0.178 | − 0.042 | 0.840 | 0.460 | 0.295 | 0.162 |
MX1 | 0.071 | 0.732 | − 0.142 | 0.489 | 0.019 | 0.926 | 0.897 | 0.073 | 0.736 |
POT1 | − 0.028 | 0.891 | − 0.041 | 0.842 | − 0.306 | 0.129 | 0.510 | − 0.075 | 0.727 |
Classification and overrepresentation analysis
PANTHER classification pathways | Number of genes | Over-/under-represented (±) | Fold enrichment | P value | ||
---|---|---|---|---|---|---|
Reference lista | Target listb | Expectedc | ||||
Apoptosis signaling pathway (P00006) | 119 | 10 | 1.68 | + | 5.96 | 0.00154 |
JAK/STAT signaling pathway (P00038) | 17 | 4 | 0.24 | + | 16.69 | 0.00210 |
Integrin signalling pathway (P00034) | 194 | 11 | 2.73 | + | 4.02 | 0.01920 |
Angiogenesis (P00005) | 174 | 10 | 2.45 | + | 4.08 | 0.03500 |