Background
Methods
Animal treatments and sample collection
Progesterone assay
Isolation total RNA and reverse transcription
miRNA Profiling and expression analysis
Target prediction and pathway analysis
Exosome isolation
Immunoprecipitation Ago2 protein complex
Western blot analysis
Individual qRT-PCR Assays
Statistical analysis
Results
Progesterone profile of experimental heifers
Detection of miRNAs in bovine follicular fluid and blood plasma
Differential expression of extracellular miRNAs in follicular fluid from hyperstimulated and unstimulated heifers
Follicular fluid (Hyp vs. Unst) | Blood plasma (Hyp vs. Unst) | ||||
---|---|---|---|---|---|
MicroRNA Name | Fold change |
P value | MicroRNA Name | Fold change |
P value |
miR-643 | 51.36 | 0.032678 | miR-708-3p | 8.15 | 0.038772 |
miR-212 | 40.19 | 0.007121 | miR-26b-3p | 4.92 | 0.031749 |
miR-148b* | 33.13 | 0.009156 | miR-598 | 3.69 | 0.002565 |
miR-548j | 23.84 | 0.031322 | miR-423-3p | 2.77 | 0.045389 |
miR-132 | 22.45 | 0.033763 | miR-130b-5p | 2.73 | 0.022855 |
miR-224* | 13.98 | 0.008991 | miR-576-5p | 2.69 | 0.024159 |
miR-129-3p | 11.4 | 0.005949 | miR-221 | 2.66 | 0.016328 |
miR-33b | 10.57 | 0.000110 | miR-1468 | 2.63 | 0.016305 |
miR-202* | 6.53 | 0.000805 | miR-18a-3p | 2.55 | 0.031849 |
miR-155* | 6.51 | 0.039305 | miR-181c | 2.40 | 0.033951 |
miR-374a | 5.2 | 0.040696 | let-7 g | 2.00 | 0.008124 |
miR-1207-5p | 5.11 | 0.000203 | miR-103 | 2.29 | 0.002404 |
miR-182 | 4.81 | 0.047938 | miR-34a | 2.13 | 0.023235 |
miR-103 | 4.49 | 0.006078 | miR-125b | −3.07 | 0.013367 |
let-7 g | 4.23 | 0.012259 | miR-410 | −3.2 | 0.038659 |
miR-106b | 3.96 | 0.034579 | miR-127-3p | −3.22 | 0.000817 |
miR-191 | 3.95 | 0.021379 | miR-494 | −3.23 | 0.008614 |
miR-542-5p | 3.78 | 0.005687 | miR-147 | −3.63 | 0.047214 |
miR-505* | 3.57 | 0.006276 | miR-99a* | −5.54 | 0.044064 |
miR-550a | 3.19 | 0.046925 | miR-134 | −7.03 | 0.043678 |
miR-107 | 3.03 | 0.034726 | miR-153 | −7.21 | 0.006251 |
miR-595 | 2.78 | 0.030403 | |||
miR-33a* | 2.74 | 0.042360 | |||
miR-374b | 2.65 | 0.038757 | |||
miR-30b | 2.62 | 0.039581 | |||
miR-31 | 2.54 | 0.029654 | |||
miR-106b* | 2.43 | 0.005030 | |||
miR-940 | 2.37 | 0.014177 | |||
miR-495 | 2.27 | 0.010936 | |||
miR-494 | 2.06 | 0.046091 | |||
miR-300 | −2.08 | 0.020863 | |||
miR-24 | −2.09 | 0.000273 | |||
miR-452 | −2.15 | 0.020850 | |||
miR-151-5p | −2.22 | 0.033121 | |||
miR-320a | −2.58 | 0.027659 | |||
miR-125a-3p | −2.78 | 0.008997 | |||
miR-99a | −2.94 | 0.048804 | |||
miR-449a | −2.97 | 0.018677 | |||
miR-206 | −3.19 | 0.010009 | |||
miR-23a | −3.26 | 0.007032 | |||
miR-27b | −3.44 | 0.013330 | |||
miR-139-5p | −3.46 | 0.006195 | |||
miR-125b-2* | −3.6 | 0.029345 | |||
miR-378 | −3.72 | 0.017333 | |||
miR-125b | −3.86 | 0.017793 | |||
miR-141 | −4.03 | 0.038193 | |||
miR-23b | −4.11 | 0.033330 | |||
miR-190 | −4.2 | 0.004785 | |||
miR-382 | −4.45 | 0.035549 | |||
miR-92a | −4.53 | 0.007967 | |||
miR-145 | −4.78 | 0.000015 | |||
miR-361-5p | −5.25 | 0.029699 | |||
miR-200a* | −6.41 | 0.040837 | |||
miR-200c | −6.48 | 0.048195 | |||
miR-659 | −8.42 | 0.037679 | |||
miR-877 | −12.16 | 0.009853 | |||
miR-100 | −14.02 | 0.004324 |
Expression profile of circulatory miRNAs in blood plasma derived from hyperstimulated vs. unstimulated heifers
Cluster of miRNAs affected by ovarian hyperstimulation in follicular fluid and blood plasma
miRNA target prediction and pathway analysis
Pathway | miRNAs involved |
P value | Top genes |
---|---|---|---|
Overexpressed miRNAs (hyp vs. unst) | |||
TGF-beta signaling pathway | miR-106b, −132, −148b-5p, −182, −212, −374a, −548j | 1.75E-07 | TGFBR2, TGFB2, SMAD2, SMAD3, SMAD4, BMPR2 |
Axon guidance | miR-22-5p, −30b, −31, −33a-3p, −182, −132, −550a | 2.85E-06 | EFNB1, DCC, EPHB4, EPHA3, PLXNA1, PAK4 |
MAPK signaling pathway | miR-30b, −106b, −132, −182, −212, −548j, −202-5p | 6.43E-05 | MAP3K1, MAP3K5, KRAS, MRAS, GRB2, FGF7 |
Endocytosis | miR-33a-3p, −106b, −182, −374a, −374b, −202-5p | 3.83E-04 | RAB11FIP4, RAB11FIP2, EEA1, IGF1R, EPS15, EPN |
Colorectal cancer | miR-30b, −33a-3p, −106b, −132, −212, −384, −494 | 5.94E-04 | SOS1, FZD3, SMAD2, DCC, MAPK1, BAX |
Pathways in cancer | miR-33a-3p, −107, −132, −212, −494, −495, −548j | 5.95E-04 | E2F1, FGF18, WNT16, FGF7, PTEN, MITF |
Wnt signaling pathway | miR-132, −212, −33a-3p, −494, −940, −495, −548j, −107 | 6.73E-04 | LRP5, LRP6, TCF7, PLCB4, DVL3, WNT1, WNT5A |
Neurotrophin signaling pathway | miR-106b, −30b, −940, −182, −212, −107, | 0.001267 | PIK3R2, NTRK2, NTRK3, RPS6KA6, IRS1, RAC1 |
Oocyte meiosis | miR-212, −132, −940, 495, −595, −107 | 0.003369 | CDC27, CPEB1, PRKACB, FBXW11, MAPK1, RPS6KA3 |
GnRH signaling pathway | miR-940, −495 | 0.007085 | PRKCA, SRC, MAP3K2, MAPK14, GRB2, MAP3K3 |
Underexpressed miRNAs (Hyp vs. Unst) | |||
Pathways in cancer | miR-659, −141, −190, −449a, −200c, −361-5p, −145 | 9.51E-12 | KRAS, PTEN, VEGFA, STAT5B, MITF, BCL2 |
MAPK signaling pathway | miR-23a, −23b, −141, −145, −200c, −92a, −125b, −24 | 2.94E-11 | MKNK2, MEF2C, FGF, SOS1, RAS, MAerkPK2 |
Wnt signaling pathway | miR-125b, −449a, −302e, −145, −23a, −29b-2-5p, −659, −200c | 2.76E-08 | LRP5, LRP6, TCF7, PLCB4, DVL3, WNT1, WNT5A |
ErbB signaling pathway | miR-23a, −23b, −125b, −206, −302e, −513c | 3.19E-07 | ERBB4, GRB2, SHC1, PIK3R3, PIK3CD, AKT3 |
Colorectal cancer | miR-659, 23a, −23b, −141, −190, −449a, −200c, −361-5p, −145 | 4.48E-07 | DCC, APPL1, TGFBR2, SMAD3, SMAD4, APC |
Axon guidance | miR-23a, −23b, −200c, −145 | 7.23E-07 | PLXNC1, EFNB2, PAK4, DCC, SEMA6A, PAK7, MET |
Neurotrophin signaling pathway | miR-302e, −361-5p, −452, −525-5p, −449a, −659 | 1.82E-06 | NTRK2, NGFR, KRAS, MAP3K1, PIK3R3, PLCG1 |
Renal cell carcinoma | miR-659, −141, −190, −449a, −200c, −361-5p, −145 | 3.75E-05 | GAB1, MET, SOS1, GRB2, VEGFA, NRAS |
Focal adhesion | miR-24, −27b, −125a-3p, −139-5p, −206, −200c | 5.04E-05 | ITGB3, PXN, SHC3, ACTB, SRC, PAK2 |
Regulation of actin cytoskeleton | miR-200c, −145, −27b, −92a, −300, −206 | 8.29E-05 | RAC1, ROCK2, GIT1, PIK3R3, ITGA3, LIMK1 |
Pathway name | miRNA involved |
P value | Top genes |
---|---|---|---|
Overexpressed miRNAs (hyp vs. unst) | |||
Pathways in cancer | let-7 g, miR- 20b-3p, −22, −26b-3p, −34a, −221, −181c | 1.44E-09 | AKT3, PIK3R3, RASSF1, KRAS, MEK, DCC |
Wnt signaling pathway | let-7 g, miR-221, −32 | 4.04E-08 | WNT1, SMAD2, DKK2, DVL3, VANGL2, CCND1 |
Neurotrophin signaling pathway | let-7 g, miR-22, −181c, −221 | 6.73E-08 | NTRK2, IRS2, AKT3, NGF, PIK3CD, NRAS |
Axon guidance | miR-34a, −181c, −221 | 2.19E-07 | PLXNC1, DPYSL2, PAK1, MET, KRAS, EFNB1 |
Endocytosis | miR-22, −34a, −130b-3p, −576-5p | 1.23E-06 | TGFBR1, RAB5A, RAB11FIP4, PDCD6IP, VPS37A |
MAPK signaling pathway | let-7 g, miR-22, −32, −34a, −221 | 2.69E-06 | RRAS, MAP3K1, MAPK1, MAPK9, SRF, MAP2K4 |
Colorectal cancer | let-7 g, miR-22, −26b-3p, −24-2-5p | 4.59E-06 | DCC, TGFBR1, KRAS, MAP2K1, AKT3, CASP3 |
Chronic myeloid leukemia | miR-22, −181c, −221 | 4.98E-06 | NRAS, AKT2, BCR, MAPK1, GAB2, BCL2L1 |
TGF-beta signaling pathway | miR-26b-3p, −221, −22, −32 | 9.63E-06 | ACVR2A, TGFBR2, SMAD2, SMAD4, BMP7 |
ErbB signaling pathway | miR-22, −181c, −221 | 2.96E-05 | ERBB4, GAB1, MAP2K1, PAK4, PIK3CD |
Underexpressed miRNAs (Hyp vs. Unst) | |||
Pathways in cancer | miR-125b, −153, −410, −494 | 9.51E-12 | DCC, GRB2, SMAD2, SOS1, E2F3 |
MAPK signaling pathway | mir-125b, −153, −494 | 2.94E-11 | MEF2C, MAPKAPK2, MAP3K1, TGFB2, FGFR2 |
Wnt signaling pathway | miR-147, −153, −410, −494 | 2.76E-08 | LRP6, WNT5A, DVL3, DKK2, PLCB1, ANGL2 |
ErbB signaling pathway | miR-125b, −410 | 3.19E-07 | ERBB4, SOS1, MAPK1, NRG3, GAB1, MAP2K7 |
Colorectal cancer | miR-125b, −410, −153, −494 | 4.48E-07 | DCC, BCL2, SMAD4, RAF1, SMAD2, PIK3R3 |
Axon guidance | miR-147, −153, −410, −494 | 7.23E-07 | PLXNA2, ROCK2, EFNA3, NFAT5, MAPK1 |
Neurotrophin signaling pathway | miR-125b, −134, −147, −494 | 1.82E-06 | NTF3, MAP3K1, SOS1, SORT1, BCL2 |
Melanogenesis | miR-134, −99a-3p | 4.52E-06 | MAPK1, WNT5A, KRAS, GNAI3, CREB1 |
Melanoma | miR-125b, −494 | 1.70E-05 | E2F2, FGFR1, IGF1R, FGF7, RAF1 |
Prostate cancer | miR-125b, −147, −153, −410, −494 | 1.82E-05 | FGFR2,, IGF1R, MAPK1, IGF1, BCL2 |