Background
Methods
Tissue collection and cell culture
Determination of E2 and P4 concentrations
RNA preparation and cDNA synthesis
Quantitative Real-Time PCR
Microarray profiling and statistics
Ingenuity Pathway Analysis (IPA)
Results
Expression profiling of GC cultured at different cell densities
Transcript cluster ID | Gene symbol | Gene title | FC |
---|---|---|---|
12837074 |
TXNIP
| thioredoxin interacting protein | −79.50 |
12832821 |
PAG11
| pregnancy-associated glycoprotein 11 | −15.47 |
12793470 |
ARRDC4
| arrestin domain containing 4 | −8.13 |
12688063 |
CYP19A1
| cytochrome P450, family 19, subfamily A, polypeptide 1 | −7.02 |
12836111 |
PIK3R3
| phosphoinositide-3-kinase, regulatory subunit 3 (gamma) | −6.55 |
12850069 |
LOC512293
| G2/mitotic-specific cyclin-B1-like | −5.49 |
12703781 |
XDH
| xanthine dehydrogenase | −5.36 |
12769776 |
SLC43A2
| solute carrier family 43, member 2 | −4.91 |
12823470 |
NRG1
| neuregulin 1 | −4.87 |
12739111 |
SUSD4
| sushi domain containing 4 | −4.83 |
12893122 |
ASPN
| asporin | −4.77 |
12703820 |
FSHR
| follicle stimulating hormone receptor | −4.64 |
12780359 |
DHRS9
| dehydrogenase/reductase (SDR family) member 9 | −4.59 |
12774405 |
INHA
| inhibin, alpha | −4.53 |
12726330 |
ANO3
| anoctamin 3 | −4.20 |
12826119 |
SRGN
| serglycin | −4.09 |
12892568 |
OMD
| osteomodulin | −4.06 |
12678550 |
SST
| somatostatin | −3.90 |
12871419 |
RASL11B
| RAS-like, family 11, member B | −3.78 |
12685216 |
KCNAB1
| potassium voltage-gated channel, shaker-related subfamily, beta member 1 | −3.77 |
Transcript cluster ID | Gene symbol | Gene title | FC |
---|---|---|---|
12812382 |
HBA
| hemoglobin, alpha 2 | 53.48 |
12683283 |
AHSG
| alpha-2-HS-glycoprotein | 36.57 |
12894529 |
LOXL2
| lysyl oxidase-like 2 | 19.01 |
12718011 |
TGM2
| transglutaminase 2 (C polypeptide, protein-glutamine-gamma-glutamyltransferase) | 17.07 |
12864766 |
KRT18
| keratin 18 | 16.63 |
12803191 |
NRN1
| neuritin 1 | 13.97 |
12854172 |
UPP1
| uridine phosphorylase 1 | 13.77 |
12793341 |
EGLN3
| egl nine homolog 3 (C. elegans) | 12.82 |
12893064 |
IL33
| interleukin 33 | 12.59 |
12856851 |
KRT8
| keratin 8 | 12.02 |
12889017 |
LPL
| lipoprotein lipase | 9.74 |
12830373 |
CDKN1C
| cyclin-dependent kinase inhibitor 1C (p57, Kip2) | 9.63 |
12688416 |
CHAC1
| ChaC, cation transport regulator homolog 1 (E. coli) | 9.56 |
12698536 |
IL1RN
| interleukin 1 receptor antagonist | 8.75 |
12817678 |
SYT17
| synaptotagmin XVII | 8.37 |
12860539 |
SLC38A4
| solute carrier family 38, member 4 | 7.67 |
12748152 |
CLDN5
| claudin 5 | 7.05 |
12703992 |
PTGES
| prostaglandin E synthase | 7.05 |
12876752 |
LOX
| lysyl oxidase | 7.04 |
12721639 |
NCALD
| neurocalcin delta | 6.84 |
Re-evaluation of microarray data by qPCR and identification of genes regulated in vivo by LH and in vitro by plating density
Gene symbol | Gene name | FC qRT-PCR | FC microarray | Correlation coefficient |
---|---|---|---|---|
CYP19A1
| cytochrome P450, family 19, subfamily A, polypeptide 1, aromatase | −11.09 | −7.02 | 0.99 |
CYP11A1
| cytochrome P450, family 11, subfamily A, polypeptide 1 | 1.65 | 1.24a
| 0.98 |
HSD3B1
| hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta | −2.43 | −1.2a
| 0.98 |
FSHR
| follicle stimulating hormone receptor | −5.97 | −4.64 | 0.99 |
LHCGR
| luteinizing hormone/choriogonadotropin receptor | −4.12 | −2.35 | 0.98 |
PTGS2
| prostaglandin-endoperoxide synthase 2 (prostaglandin G/H synthase) | 1.09a
| 1.27a
| 0.78 |
RGS2
| regulator of G-protein signaling 2 | 4.84 | 3.3 | 0.99 |
VNN2
| vanin 2 | 7.75 | 5.88 | 0.99 |
PTX3
| pentraxin 3, long | 3.95 | 3.02 | 0.96 |
PCNA
| proliferating cell nuclear antigen | −1.16a
| −1.18a
| 0.83 |
CCND2
| cyclin D2 | −1.69 | −1.37a
| 0.96 |
Gene symbol | Gene title | FC in vitro | FC in vivo | corr. r
|
p-value |
---|---|---|---|---|---|
P4HA3
| prolyl 4-hydroxylase, alpha polypeptide III | 1.7 | 75.8 | 0.997 | 0.00001 |
ITGA4
| integrin, alpha 4 (antigen CD49D, alpha 4 subunit of VLA-4 receptor) | 1.9 | 7.2 | 0.995 | 0.00004 |
WWC2
| WW and C2 domain containing 2 | 1.5 | 3.9 | 0.995 | 0.00004 |
TACC3
| transforming, acidic coiled-coil containing protein 3 | −1.7 | −5.4 | 0.995 | 0.00004 |
ATAD2
| ATPase family, AAA domain containing 2 | −1.8 | −8.1 | 0.994 | 0.00005 |
INHBA
| inhibin, beta A | −2.4 | −29.5 | 0.994 | 0.00005 |
CAPG
| capping protein (actin filament), gelsolin-like | 1.7 | 7.3 | 0.994 | 0.00005 |
LOC784007
| uncharacterized LOC784007; SLAM family member 9-like | −2.0 | −21.8 | 0.993 | 0.00007 |
ITPKA
| inositol-trisphosphate 3-kinase A | 2.2 | 6.7 | 0.993 | 0.00007 |
QSOX1
| quiescin Q6 sulfhydryl oxidase 1 | 1.6 | 5.4 | 0.992 | 0.00009 |
NDRG1
| N-myc downstream regulated 1 | 4.3 | 63.8 | 0.992 | 0.00010 |
SAT1
| spermidine/spermine N1-acetyltransferase 1 | 1.6 | 15.0 | 0.991 | 0.00013 |
CDCA3
| cell division cycle associated 3 | −1.6 | −11.0 | 0.989 | 0.00017 |
RGS2
| regulator of G-protein signaling 2, 24 kDa | 3.3 | 194.0 | 0.989 | 0.00020 |
HEG1
| HEG homolog 1 (zebrafish) | −2.1 | −29.7 | 0.985 | 0.00035 |
PTX3
| pentraxin 3, long | 3.0 | 643.9 | 0.958 | 0.00257 |
SRGN
| serglycin | −4.1 | −29.9 | 0.924 | 0.00841 |
ADAMTS1
| ADAM metallopeptidase with thrombospondin type 1 motif, 1 | 1.9 | 28.5 | 0.967 | 0.00166 |
CYP19A1
| cytochrome P450, family 19, subfamily A, polypeptide 1 | −7.0 | −397.6 | 0.912 | 0.01140 |
HSD11B1
| hydroxysteroid (11-beta) dehydrogenase 1 | 2.4 | 14.5 | 0.898 | 0.01493 |
FSHR
| follicle stimulating hormone receptor | −4.6 | −4.1 | 0.896 | 0.01555 |
LHCGR
| luteinizing hormone/choriogonadotropin receptor | −2.4 | −7.9 | 0.811 | 0.05005 |
AHSG
| alpha-2-HS-glycoprotein | 36.6 | 152.3 | 0.809 | 0.05110 |
VNN2
| vanin 2 | 5.9 | 116.0 | 0.749 | 0.08659 |
CDKN1C
| cyclin-dependent kinase inhibitor 1C (p57, Kip2) | 9.6 | 24.4 | 0.718 | 0.10777 |
Pathway analysis and upstream regulators
Ingenuity canonical pathways | -log(p-value) |
p-value | Ratio | z-scorea
| Number of affected molecules | Total number of moleculesb
|
---|---|---|---|---|---|---|
AMPK Signaling | 4.00 | 0.0001 | 0.183 | −0.471 | 30 | 164 |
Hepatic Fibrosis/Hepatic Stellate Cell Activation | 3.76 | 0.0002 | 0.180 | 29 | 161 | |
Acetate Conversion to Acetyl-CoA | 3.55 | 0.0003 | 0.800 | 4 | 5 | |
Ovarian Cancer Signaling | 3.45 | 0.0004 | 0.190 | 23 | 121 | |
cAMP-mediated signaling | 3.25 | 0.0006 | 0.162 | 1.257 | 32 | 197 |
Mitotic Roles of Polo-Like Kinase | 3.08 | 0.0008 | 0.236 | 13 | 55 | |
Glioma Signaling | 3.04 | 0.0009 | 0.198 | 0.258 | 18 | 91 |
Cell Cycle Control of Chromosomal Replication | 2.87 | 0.0013 | 0.308 | 8 | 26 | |
Xenobiotic Metabolism Signaling | 2.79 | 0.0016 | 0.151 | 33 | 218 | |
LXR/RXR Activation | 2.69 | 0.0020 | 0.181 | 0.775 | 19 | 105 |
Calcium Signaling | 2.62 | 0.0024 | 0.161 | 1.698 | 25 | 155 |
Eumelanin Biosynthesis | 2.59 | 0.0026 | 0.750 | 3 | 4 | |
FXR/RXR Activation | 2.50 | 0.0032 | 0.174 | 19 | 109 | |
ATM Signaling | 2.50 | 0.0032 | 0.214 | −1.265 | 12 | 56 |
Ethanol Degradation IV | 2.46 | 0.0035 | 0.333 | 6 | 18 | |
LPS/IL-1 Mediated Inhibition of RXR Function | 2.45 | 0.0035 | 0.151 | 0.378 | 28 | 185 |
Glioma Invasiveness Signaling | 2.43 | 0.0037 | 0.211 | 0.577 | 12 | 57 |
GADD45 Signaling | 2.33 | 0.0047 | 0.316 | 6 | 19 | |
Superpathway of Cholesterol Biosynthesis | 2.31 | 0.0049 | 0.280 | 7 | 25 | |
Oxidative Ethanol Degradation III | 2.26 | 0.0055 | 0.357 | 5 | 14 |
Category |
p-valuea
| Number of molecules affected | Total number of moleculesb
|
---|---|---|---|
Cellular Growth and Proliferation | 3.8E-20 - 8.8E-05 | 563 | 5452 |
Cell Death and Survival | 2.47E-15 - 9.69E-05 | 483 | 4545 |
Cell Cycle | 6.45E-15 - 7.83E-05 | 232 | 2255 |
Cellular Assembly and Organization | 6.45E-15 - 7.34E-05 | 260 | 4244 |
DNA Replication, Recombination, and Repair | 6.45E-15 - 8.49E-05 | 128 | 1979 |
Cellular Movement | 1.01E-13 - 8.74E-05 | 331 | 3043 |
Cellular Development | 3.58E-12 - 8.8E-05 | 528 | 5390 |
Lipid Metabolism | 4.8E-12 - 7.98E-05 | 238 | 2105 |
Small Molecule Biochemistry | 4.8E-12 - 7.98E-05 | 258 | 4000 |
Cell Morphology | 4.52E-11 - 9.41E-05 | 376 | 4268 |