Table 1. Differentially expressed miRNAs in the screening phase.
miRNA Fold change Mean fold
Pool 1 Pool 2 Pool 3
miR-140-5p 3.65 2.90 2.44 2.99
miR- 144-5p 1.76 1.55 1.82 1.71
miR- 155-5p 2.48 1.98 1.83 2.10
miR- 17-5p 2.00 2.44 2.67 2.37
miR- 200a-3p 1.88 2.34 1.90 2.04
miR- 210 3.01 1.97 1.87 2.28
miR- 223-5p 4.12 3.67 6.77 4.85
miR- 28-3p 1.74 2.09 1.64 1.81
miR- 301a-3p 2.33 2.67 1.75 2.25
miR- 409-3p 2.07 2.88 2.23 2.39
miR- 425-5p 3.01 4.59 2.76 3.45
miR- 497-5p 1.62 1.90 1.57 1.70
miR- 574-5p 2.02 1.59 1.77 1.79
miR- 92b-3p 7.01 5.77 5.23 6.00
miR- 10b-5p 5.35 4.22 5.98 5.18
miR- 141-3p 2.54 3.01 1.87 2.47
miR- 146b-5p 2.36 2.03 1.89 2.09
miR- 18a-3p 4.98 2.78 3.66 3.81
miR- 200c-3p 3.64 2.99 2.76 3.13
miR- 21-5p 3.12 2.87 4.01 3.33
miR- 24-3p 1.78 2.05 2.01 1.95
miR- 29a-5p 2.69 2.49 1.95 2.38
miR- 34a-5p 3.57 3.28 3.00 3.28
miR- 25-3p 1.73 1.99 2.44 2.05
miR- 29b-2-5p 3.89 4.02 1.76 3.22
miR- 361-3p 1.83 2.38 1.75 1.99
miR- 584-5p 6.96 5.57 6.32 6.28
miR- 99a-5p 1.68 1.90 2.33 1.97
miR- 133a 3.45 2.98 4.02 3.48
miR- 144-3p 2.56 2.48 2.65 2.56
miR- 151a-3p 1.76 2.09 2.57 2.14
miR- 16-2-3p 3.37 2.81 2.32 2.83
miR- 19b-3p 1.63 1.87 1.99 1.83
miR- 20a-5p 3.57 3.71 1.97 3.08
miR- 221-3p 3.79 5.02 4.22 4.34
Note: miRNAs in bold were identified after the training phase.
Table 2. The cycle threshold (Ct) values of miR-23a and miR-451 in the 24 serum LA samples and 24 NC samples.
Sample miR-23a Ct values miR-451 ΔCt
LA1 31.303 28.803 2.500
LA2 30.257 25.720 4.537
LA3 29.520 27.593 1.927
LA4 30.410 27.023 3.387
LA5 29.780 27.600 2.180
LA6 30.645 28.610 2.035
LA7 30.453 28.970 1.483
LA8 30.200 29.060 1.140
LA9 31.247 29.259 1.997
LA10 30.793 28.963 1.830
LA11 31.600 32.487 -0.887
LA12 31.580 29.177 2.403
LA13 33.215 29.623 3.592
LA14 32.913 29.613 3.300
LA15 31.330 29.807 1.523
LA16 32.715 30.077 2.638
LA17 29.947 26.033 3.913
LA18 29.870 28.620 1.250
LA19 29.503 29.027 0.477
LA20 30.983 29.730 1.253
LA21 30.290 26.303 3.987
LA22 31.310 28.820 2.490
LA23 30.627 28.503 2.123
LA24 28.667 24.360 4.307
NC1 29.317 27.007 2.310
NC2 30.037 28.567 1.470
NC3 30.130 29.087 1.043
NC4 31.010 29.820 1.190
NC5 30.820 30.380 0.440
NC6 30.855 31.815 -0.960
NC7 30.625 32.437 -1.817
NC8 30.785 31.127 -0.342
NC9 32.067 28.575 3.492
NC10 30.790 29.467 1.323
NC11 32.190 30.910 1.280
NC12 31.667 29.057 2.610
NC13 32.427 31.023 1.403
NC14 31.540 29.830 1.710
NC15 32.487 32.230 0.257
NC16 32.497 31.650 0.847
NC17 30.440 27.687 2.753
NC18 30.427 27.203 3.223
NC19 29.667 28.650 1.017
NC20 30.655 27.127 3.528
NC21 31.347 29.700 1.647
NC22 30.687 27.900 2.787
NC23 30.013 28.307 1.707
NC24 29.983 27.437 2.547
ΔCt = Ct
(miR-23a)- Ct
(miR-451); LA: Lung adenocarcinoma; NC: Normal control.
Table 3. Expression levels of the 4 miRNAs in the peripheral serum of patients in comparison with normal controls.
miRNA
Training stage Testing stage Validation stage Combined
FC Pvalue FC Pvalue FC Pvalue FC Pvalue
miR-133a-3p 6.741 0.161 11.870 < 0.001 16.442 < 0.001 11.908 < 0.001 miR-584-5p 3.633 0.061 3.101 < 0.001 3.752 0.019 3.359 < 0.001 miR-10b-5p 6.569 0.458 5.044 < 0.001 9.621 0.004 6.823 < 0.001 miR-221-3p 2.232 0.061 2.042 < 0.001 2.873 0.005 2.356 < 0.001
FC: Fold change.
Table 4. Comparison of serum miRNA expression among lung adenocarcinoma subgroups.
Variables miR-133a-3p miR-584-5p miR-10b-5p miR-221-3p
TNM stage 0.744 0.971 0.526 0.532
Lymph node 0.391 0.115 0.032 0.267
Differentiation 0.423 0.058 0.171 0.279
Location 0.482 0.435 0.283 0.237
Histologic Classification 0.266 0.178 0.412 0.383
Tumor Size 0.189 0.092 0.361 0.092
Presented as the P-values of Mann-WhitneyUtest or Kruskal-Wallis rank test;P> 0.05 means there is no reason to reject the null hypothesis that miRNA expression levels are the same among the subtypes; TNM: The T(tumor) N(nodes) M(metastases) classification of malignant tumor.
Table 5. KEGG pathway analysis for miR-584-5p, miR-10b-5p and miR-221-3p.
miRNA Pathway P value
miR-584-5p Prion diseases < 0.001
ECM-receptor interaction < 0.001
miR-10b-5p Fatty acid biosynthesis < 0.001
Viral carcinogenesis < 0.001
Fatty acid metabolism < 0.001
Chronic myeloid leukemia < 0.001
Central carbon metabolism in cancer 0.002
Non-small cell lung cancer 0.007
Glycosphingolipid biosynthesis - lacto and neolacto series 0.016
Pyrimidine metabolism 0.021
Cell cycle 0.022
p53 signaling pathway 0.028
miR-221-3p Viral carcinogenesis < 0.001
Fatty acid elongation 0.002
Hippo signaling pathway 0.002
p53 signaling pathway 0.007
Proteoglycans in cancer 0.012
Chronic myeloid leukemia 0.013
Protein processing in endoplasmic reticulum 0.02
Leukocyte transendothelial migration 0.023
Transcriptional misregulation in cancer 0.023
Cell cycle 0.027
MicroRNAs in cancer 0.046
KEGG: Kyoto encyclopedia of genes and genomes.
Table 6. GO category analysis for miR-584-5p, miR-10b-5p and miR-221-3p.
miRNA GO category Pvalue
miR-584-5p organelle < 0.001
cellular nitrogen compound metabolic process < 0.001
RNA binding < 0.001
ion binding < 0.001
gene expression 0.001
biological_process 0.001
poly(A) RNA binding 0.001
cytosol 0.004
viral process 0.004
cellular_component 0.004
ATP-dependent RNA helicase activity 0.009
symbiosis, encompassing mutualism through parasitism 0.010
enzyme binding 0.000
biosynthetic process 0.018
osteoblast differentiation 0.025
catabolic process 0.032
nucleobase-containing compound catabolic process 0.033
membrane organization 0.041
miR-10b-5p organelle < 0.001
cellular nitrogen compound metabolic process < 0.001
gene expression < 0.001
biosynthetic process < 0.001
nucleoplasm < 0.001
cytosol < 0.001
enzyme binding < 0.001
symbiosis, encompassing mutualism through parasitism < 0.001
protein complex < 0.001
molecular_function < 0.001
viral process < 0.001
cellular_component < 0.001
nucleic acid binding transcription factor activity < 0.001
biological_process < 0.001
RNA binding < 0.001
response to stress < 0.001
cellular protein modification process < 0.001
ion binding < 0.001
mRNA metabolic process < 0.001
mitotic cell cycle < 0.001
RNA metabolic process < 0.001
catabolic process < 0.001
small molecule metabolic process < 0.001
transcription, DNA-templated < 0.001
protein binding transcription factor activity < 0.001
poly(A) RNA binding < 0.001
cellular heat acclimation < 0.001
cellular protein metabolic process < 0.001
macromolecular complex assembly < 0.001
nucleobase-containing small molecule interconversion < 0.001 RNA polymerase II core promoter sequence-specific DNA binding < 0.001 transcription initiation from RNA polymerase II promoter < 0.001 positive regulation of erythrocyte differentiation < 0.001
cellular component assembly < 0.001
Fc-epsilon receptor signaling pathway < 0.001
neurotrophin TRK receptor signaling pathway < 0.001
regulation of transcription from RNA polymerase II promoter < 0.001
negative regulation of cell proliferation < 0.001
transcription factor binding < 0.001
positive regulation of transcription, DNA-templated < 0.001
viral life cycle 0.001
nucleobase-containing compound catabolic process 0.001
nucleobase-containing small molecule metabolic process 0.001
G1/S transition of mitotic cell cycle 0.001
mRNA splicing, via spliceosome 0.001
regulation of ubiquitin-protein ligase activity involved in mitotic cell
cycle 0.001
negative regulation of transcription, DNA-templated 0.001
transcription from RNA polymerase II promoter 0.001
ribonucleoprotein complex assembly 0.001
alternative mRNA splicing, via spliceosome 0.003
histone acetyltransferase binding 0.003
DNA damage response, signal transduction by p53 class mediator
resulting in cell cycle arrest 0.003
positive regulation of cell cycle arrest 0.003
RNA splicing, via transesterification reactions 0.004
virus receptor activity 0.005
core promoter sequence-specific DNA binding 0.006
nuclear-transcribed mRNA catabolic process, deadenylation-dependent
decay 0.007
protein targeting 0.007
anaphase-promoting complex-dependent proteasomal
ubiquitin-dependent protein catabolic process 0.007
positive regulation of ubiquitin-protein ligase activity involved in mitotic
cell cycle 0.007
cell death 0.007
protein N-terminus binding 0.008
RNA polymerase II transcription factor binding transcription factor
activity involved in positive regulation of transcription 0.008
circadian rhythm 0.008
cyclin-dependent protein kinase holoenzyme complex 0.009
negative regulation of inclusion body assembly 0.009
positive regulation of protein insertion into mitochondrial membrane
involved in apoptotic signaling pathway 0.011
ligase activity 0.011
negative regulation of transcription from RNA polymerase II promoter 0.011
viral transcription 0.012
HMG box domain binding 0.013
negative regulation of osteoblast differentiation 0.013
intrinsic apoptotic signaling pathway 0.015
cellular lipid metabolic process 0.015
nuclear chromatin 0.016
RNA polymerase II core promoter proximal region sequence-specific DNA binding transcription factor activity involved in positive regulation
of transcription 0.017
negative regulation of cell growth 0.020
hexose transport 0.021
DNA recombinase assembly 0.026
cyclin binding 0.026
muscle cell differentiation 0.026
negative regulation of oligodendrocyte differentiation 0.026 dosage compensation by inactivation of X chromosome 0.028
mRNA processing 0.028
transcription-coupled nucleotide-excision repair 0.028
phosphatidylinositol-mediated signaling 0.028
protein binding involved in protein folding 0.029
core promoter binding 0.029
membrane organization 0.029
telomere maintenance via semi-conservative replication 0.029 negative regulation of ubiquitin-protein ligase activity involved in
mitotic cell cycle 0.031
nuclear speck 0.032
glucose transport 0.034
RNA splicing 0.035
poly(U) RNA binding 0.036
double-stranded RNA binding 0.036
cell cycle 0.036
cellular response to heat 0.038
positive regulation of extrinsic apoptotic signaling pathway in absence of
ligand 0.043
negative regulation of calcium ion-dependent exocytosis 0.044
insulin receptor signaling pathway 0.045
translational termination 0.046
cellular response to glucose starvation 0.047
cell junction organization 0.049
miR-221-3p Viral carcinogenesis < 0.001
Fatty acid elongation 0.002
Hippo signaling pathway 0.002
p53 signaling pathway 0.007
Proteoglycans in cancer 0.012
Chronic myeloid leukemia 0.013
Protein processing in endoplasmic reticulum 0.020
Leukocyte transendothelial migration 0.023
Transcriptional misregulation in cancer 0.023
Cell cycle 0.027
MicroRNAs in cancer 0.046
