Background
Results
Virus infection and observation of cytopathic effect
MiRNA microarray analysis
miR-name
|
Fold change
|
P
values
|
Target sequence (5′ to 3′)
|
---|---|---|---|
Up-regulated miRNAs
| |||
hsa-miR-4800-3p | 28.83 | 4.19E-03 | CAUCCGUCCGUCUGUCCAC |
hsa-miR-4530 | 12.00 | 4.64E-03 | CCCAGCAGGACGGGAGCG |
hsa-miR-4492 | 9.61 | 7.36E-03 | GGGGCUGGGCGCGCGCC |
hsa-miR-4507 | 8.88 | 3.76E-02 | CUGGGUUGGGCUGGGCUGGG |
hsa-miR-3620-5p | 8.86 | 1.18E-02 | GUGGGCUGGGCUGGGCUGGGCC |
hsa-miR-4505 | 8.72 | 6.33E-03 | AGGCUGGGCUGGGACGGA |
hsa-miR-1587 | 8.54 | 2.03E-02 | UUGGGCUGGGCUGGGUUGGG |
hsa-miR-7108-5p | 8.19 | 2.11E-02 | GUGUGGCCGGCAGGCGGGUGG |
hsa-miR-6791-5p | 6.14 | 2.63E-02 | CCCCUGGGGCUGGGCAGGCGGA |
hsa-miR-6125 | 5.71 | 2.61E-02 | GCGGAAGGCGGAGCGGCGGA |
hsa-miR-494-3p | 5.49 | 3.58E-02 | UGAAACAUACACGGGAAACCUC |
hsa-miR-1973 | 5.11 | 4.13E-04 | ACCGUGCAAAGGUAGCAUA |
hsa-miR-6729-5p | 4.96 | 2.12E-02 | UGGGCGAGGGCGGCUGAGCGGC |
hsa-miR-638 | 4.61 | 1.17E-02 | AGGGAUCGCGGGCGGGUGGCGGCCU |
hsa-miR-3960 | 4.15 | 1.55E-02 | GGCGGCGGCGGAGGCGGGGG |
hsa-miR-6126 | 4.05 | 3.70E-02 | GUGAAGGCCCGGCGGAGA |
hsa-miR-4787-5p | 4.04 | 1.08E-02 | GCGGGGGUGGCGGCGGCAUCCC |
hsa-miR-8072 | 4.04 | 2.07E-02 | GGCGGCGGGGAGGUAGGCAG |
hsa-miR-6085 | 3.90 | 1.49E-02 | AAGGGGCUGGGGGAGCACA |
hsa-miR-6779-5p | 3.85 | 1.75E-02 | CUGGGAGGGGCUGGGUUUGGC |
hsa-miR-6087 | 3.49 | 1.02E-02 | UGAGGCGGGGGGGCGAGC |
hsa-miR-4707-5p | 3.33 | 1.81E-02 | GCCCCGGCGCGGGCGGGUUCUGG |
hsa-miR-7704 | 3.26 | 4.44E-03 | CGGGGUCGGCGGCGACGUG |
hsa-miR-3196 | 3.25 | 8.02E-03 | CGGGGCGGCAGGGGCCUC |
hsa-miR-6743-5p | 3.03 | 6.81E-03 | AAGGGGCAGGGACGGGUGGCCC |
hsa-miR-4466 | 2.94 | 1.88E-02 | GGGUGCGGGCCGGCGGGG |
hsa-miR-3665 | 2.77 | 3.44E-02 | AGCAGGUGCGGGGCGGCG |
hsa-miR-4497 | 2.71 | 1.41E-02 | CUCCGGGACGGCUGGGC |
hsa-miR-6803-5p | 2.62 | 3.88E-02 | CUGGGGGUGGGGGGCUGGGCGU |
hsa-miR-5787 | 2.24 | 2.21E-02 | GGGCUGGGGCGCGGGGAGGU |
hsa-miR-7641 | 2.09 | 2.70E-02 | UUGAUCUCGGAAGCUAAGC |
hsa-miR-4298 | 2.00 | 3.56E-02 | CUGGGACAGGAGGAGGAGGCAG |
hsa-miR-4436b-3p | 1.99 | 1.04E-02 | CAGGGCAGGAAGAAGUGGACAA |
hsa-miR-4459 | 1.74 | 8.61E-03 | CCAGGAGGCGGAGGAGGUGGAG |
hsa-miR-4488 | 1.63 | 4.31E-02 | AGGGGGCGGGCUCCGGCG |
hsa-miR-4739 | 1.45 | 1.30E-02 | AAGGGAGGAGGAGCGGAGGGGCCCU |
Down-regulated miRNAs
| |||
hsa-let-7a-5p | 0.70 | 3.40E-03 | UGAGGUAGUAGGUUGUAUAGUU |
hsa-miR-29a-3p | 0.69 | 1.20E-02 | UAGCACCAUCUGAAAUCGGUUA |
hsa-miR-127-3p | 0.67 | 4.16E-02 | UCGGAUCCGUCUGAGCUUGGCU |
hsa-miR-196a-5p | 0.59 | 3.35E-02 | UAGGUAGUUUCAUGUUGUUGGG |
hsa-miR-378c | 0.58 | 4.53E-02 | ACUGGACUUGGAGUCAGAAGAGUGG |
hsa-let-7b-5p | 0.55 | 8.67E-03 | UGAGGUAGUAGGUUGUGUGGUU |
hsa-miR-4443 | 0.32 | 1.17E-03 | UUGGAGGCGUGGGUUUU |
hsa-miR-27b-5p | 0.21 | 4.34E-02 | AGAGCUUAGCUGAUUGGUGAAC |
hsa-miR-1538 | 0.16 | 2.10E-02 | CGGCCCGGGCUGCUGCUGUUCCU |
Target genes prediction of the differentially expressed miRNAs and functional analysis
Pathway Id
|
Pathway description
|
S gene number
|
TS gene number
|
B gene number
|
TB gene number
|
P
value of Fisher’s exact test
|
---|---|---|---|---|---|---|
4144 | Endocytosis | 158 | 3220 | 181 | 4524 | 0.000000 |
4510 | Focal adhesion | 163 | 3220 | 193 | 4524 | 0.000008 |
4010 | MAPK signaling pathway | 209 | 3220 | 257 | 4524 | 0.000084 |
5410 | Hypertrophic cardiomyopathy (HCM) | 74 | 3220 | 84 | 4524 | 0.000168 |
4916 | Melanogenesis | 86 | 3220 | 100 | 4524 | 0.000344 |
4012 | ErbB signaling pathway | 73 | 3220 | 84 | 4524 | 0.000490 |
5214 | Glioma | 57 | 3220 | 64 | 4524 | 0.000509 |
5210 | Colorectal cancer | 72 | 3220 | 83 | 4524 | 0.000605 |
4512 | ECM-receptor interaction | 70 | 3220 | 81 | 4524 | 0.000917 |
4142 | Lysosome | 95 | 3220 | 113 | 4524 | 0.000946 |
5220 | Chronic myeloid leukemia | 63 | 3220 | 73 | 4524 | 0.001796 |
5223 | Non-small cell lung cancer | 47 | 3220 | 53 | 4524 | 0.002023 |
4910 | Insulin signaling pathway | 109 | 3220 | 133 | 4524 | 0.002627 |
5212 | Pancreatic cancer | 61 | 3220 | 71 | 4524 | 0.002697 |
4912 | GnRH signaling pathway | 78 | 3220 | 93 | 4524 | 0.003094 |
4722 | Neurotrophin signaling pathway | 101 | 3220 | 123 | 4524 | 0.003313 |
4130 | SNARE interactions in vesicular transport | 32 | 3220 | 35 | 4524 | 0.003616 |
4520 | Adherens junction | 62 | 3220 | 73 | 4524 | 0.004499 |
4514 | Cell adhesion molecules (CAMs) | 105 | 3220 | 129 | 4524 | 0.004825 |
5414 | Dilated cardiomyopathy | 72 | 3220 | 86 | 4524 | 0.004880 |
MiRNA expression validation by qRT-PCR
Discussion
Materials and methods
Cell culture, virus infection and observation of cytopathic effect
Total RNA extraction and quality control
MicroRNA microarray assay
Target gene prediction, GO enrichment and KEGG pathway analysis
Validation of microarray data with qPCR analysis
Primer name
|
Forward primer sequence (5′–3′)
|
Product size (bp)
|
---|---|---|
Up-regulated miRNAs
| ||
hsa-miR-4530 | CAGGACGGGAGCGAAAA | 66 |
hsa-miR-4492 | CTGGGCGCGCGCCAAA | 66 |
hsa-miR-4505 | CTGGGCTGGGACGGAAAA | 68 |
hsa-miR-6125 | GCGGAGCGGCGGAAAA | 67 |
hsa-miR-494-3p | GAAACATACACGGGAAACCTCAA | 74 |
hsa-miR-638 | GGGCGGGTGGCGGCCTAAA | 69 |
hsa-miR-6743-5p | AGGGACGGGTGGCCCA | 69 |
hsa-miR-4459 | GCGGAGGAGGTGGAGAAA | 68 |
Down-regulated miRNAs
| ||
hsa-miR-29a-3p | CACCATCTGAAATCGGTTAA | 72 |
hsa-miR-4443 | TGGAGGCGTGGGTTTTAAA | 69 |
hsa-miR-27b-5p | AGAGCTTAGCTGATTGGTGAACAA | 75 |