WGCNA co-expression network construction.
The outlier sample (GSM1940547) was removed, and the remaining gene expression profile and corresponding clinical traits of 79 preeclampsia samples in GSE75010 were finally enrolled into WGCNA analysis (Fig. 1). We selected the power of β = 6 as the soft-thresholding power to achieve the optimal scale independence (> 0.85) and mean connectivity (~ 0) (Fig. 2a-b). Similarly, checking scale free topology showed that the soft-thresholding power was suitable which ensured scale free R2 ≥ 0.85 (Fig. 2c). Finally, thirty three co-expression modules were obtained including floral white module, light green module, maroon module, thistle2 module, cyan module, dark orange module, paleturquoise module, light pink4 module ,plum2 module, light cyan module, brown4 module, dark magenta module, purple module, navajowhite2 module, grey60 module, palevioletred3 module, sky blue3 module, ivory module, pink module, yellow green module, midnight blue module, dark turquoise module, light yellow module, bisque4 module, white module, salmon4 module, steel blue module, dark slate blue module, yellow module, darkorange2 module, blue module, green module. Genes without co-expression relationship were set in grey module (Fig. 2d).
Identification of clinically significant modules and module core genes.
In order to clarify the relationship between preeclampsia clinical traits and gene modules, we performed Pearson analysis on each module and clinical traits. Genes in light green module were significantly associated with gestation duration (R = 0.31, P = 0.006) and nulliparity (R=-0.4, P = 0.0003). The genes of cyan module were significantly associated with umbilical cord diameter (R=-0.39, P = 0.0003). Moreover, genes in dark orange module were significantly associated with newborn weight z-score (R=-0.39, P = 0.0005), and were significantly associated with umbilical cord diameter (R=-0.32, P = 0.004). Genes in light cyan module were significantly associated with age (R=-0.31, P = 0.005), while genes in navajowhite2 module were significantly associated with BMI (R = 0.49, P = 0.000004). The genes in purple module were significantly associated with the mean umbilical PI (R=-0.32, P = 0.004), while the gens in pink modules were significantly associated with mean uterine PI (R = 0.38, P = 0.0005) and gestation days (R=-0.41, P = 0.0002). Furthermore, the floral white module, plum2 module, dark magenta module and yellow green module were closely related to various clinical traits (> 3), so these were regarded as the significant modules for subsequent analysis (Fig. 3). Through calculating the module membership (MM) of each gene in significant modules, 75 genes with MM > 0.8 were identified and set as module core genes (Table 1).
Table 1
Identification of module core genes
Gene | Module color |
PECAM1.1 | dark magenta |
GUCY1B3 | dark magenta |
CD34 | dark magenta |
GJC1 | dark magenta |
TCF4 | dark magenta |
PLSCR4 | dark magenta |
SPTAN1 | dark magenta |
MEF2C | dark magenta |
FLI1 | dark magenta |
EGFLAM | dark magenta |
KL | dark magenta |
ADGRL2 | dark magenta |
PCDHB14 | dark magenta |
ADAMTSL3 | dark magenta |
CYTH4 | floral white |
BTK | floral white |
CYBB | floral white |
CD53 | floral white |
CD4 | floral white |
ITGAM | floral white |
TFEC | floral white |
HNMT | floral white |
TBXAS1 | floral white |
MS4A14 | floral white |
MILR1 | floral white |
SLC7A7 | floral white |
SYK | floral white |
TMSB4X | floral white |
HCK | floral white |
TYROBP | floral white |
AOAH | floral white |
CD84 | floral white |
NCKAP1L | floral white |
CORO1A | floral white |
CTSS | floral white |
LRMP | floral white |
CD86 | floral white |
TNFSF8 | floral white |
PIK3CG | floral white |
TRPS1 | floral white |
LAIR1 | floral white |
PLEK | floral white |
PRKCB | floral white |
PTPRC | floral white |
SH3BGRL | floral white |
TLR1 | floral white |
TLR4 | floral white |
GPR65 | floral white |
C3AR1 | floral white |
FCER1G | floral white |
AIF1 | floral white |
DOCK2 | floral white |
LCP2 | floral white |
LAPTM5 | floral white |
MYO1F | floral white |
SAMHD1 | floral white |
TLR7 | floral white |
TLR8 | floral white |
ADAP2 | floral white |
ARHGAP15 | floral white |
RASSF4 | floral white |
HAVCR2 | floral white |
OLFML2B | plum2 |
SEC14L1 | plum2 |
AFF2 | plum2 |
HIP1.1 | plum2 |
KCNK3 | plum2 |
PLCG1 | plum2 |
TMEM204 | plum2 |
ATP1A1 | yellow green |
ZMYND11 | yellow green |
TMEM139 | yellow green |
THSD4 | yellow green |
FBN2 | yellow green |
SLC23A2 | yellow green |
Annotation:Genes in significant modules with module membership ≥ 0.8 were set as module core genes and showed in the list. |
Functional enrichment analysis and KEGG analysis for module core genes.
Module core genes were uploaded to the DAVID database for GO functional annotation. The top 10 results showed that the genes in key clinical significant modules were mainly enriched in adaptive immune response (BP, GO: 0002250), innate immune response (BP, GO: 0045087), integral component of plasma membrane (CC, GO: 0005887), inflammatory response (BP, GO: 0006954), plasma membrane (CC, GO: 0005886), B cell receptor signaling pathway (BP, GO: 0050853), toll-like receptor signaling pathway (BP, GO: 0002224 ), positive regulation of T cell proliferation (BP, GO: 0042102 ), MyD88-dependent toll-like receptor signaling pathway (BP, GO: 0002755), leukocyte migration (BP,GO: 0050900) (Fig. 4). The KEGG pathway enrichment analysis reflected that module core genes were enriched in pathways platelet activation, Fc gamma R-mediated phagocytosis, tuberculosis, natural killer cell mediated cytotoxicity, Fc epsilon RI signaling pathway, NF-kappa B signaling pathway, Toll-like receptor signaling pathway, Cell adhesion molecules (CAMs), phospholipase D signaling pathway and phagosome (Fig. 5).
PPI network construction and identification of hub genes.
Module core genes were uploaded to the String database for construction of PPI network (Fig. 6a). R was used to calculate the number of connections between genes, and the genes including PTPRC, TYROBP, PLEK, LCP2, HCK, ITGAM, BTK, CD86 with the top 10%degree score were obtained as hub genes (Fig. 6b). These hub genes were used further analysis.
Identification the expression of core genes between preeclampsia placentas and non-preeclampsia placentas.
Furthermore, the expression of hub genes between 79 preeclampsia placentas and 77 non-preeclampsia placentas in GSE75010 were analyzed. The results showed that the expression levels of these hub genes including PTPRC, TYROBP, PLEK, LCP2, HCK, ITGAM, BTK, CD86 were significantly decreased in the preeclampsia placentas compared with the non-preeclampsia placentas (Fig. 7).
Diagnostic efficiency analysis of hub genes.
Then, the gene expression profile GSE25906 included 23 preeclampsia placentas and 37 non- preeclampsia placentas were used to detect the diagnostic efficiency of hub genes via constructing ROC curve. The results showed that TYROBP (AUC = 0.744), PLEK (AUC = 0.783), LCP2 (AUC = 0.732), HCK (AUC = 0.669), ITGAM (AUC = 0.746) exhibited remarkable diagnostic efficiency (Fig. 8).
Immunohistochemical verification results of hub genes.
In order to verify the expression of hub genes between the two groups, we performed immunohistochemistry on 18 preeclamptic placenta tissues and 15 normal control placenta tissues. There was no difference in gestational days, age, and BMI between the two groups of pregnant women (Table 2). The results showed that these hub genes (TYROBP, PLEK, LCP2, HCK, ITGAM) have lower expression in preeclampsia than the normal group (Fig. 9).
Table 2
Clinical characteristics of two groups of pregnant women
Clinical traits | group |
Control (n = 15) | Preeclampsia (n = 18) |
gestational age, days | 260.40 ± 18.59 | 251.94 ± 23.13 |
birth weight, g | 2847.33 ± 603.83 | 2247.78 ± 749.61* |
placenta weight, g | 584.67 ± 106.90 | 493.33 ± 139.58* |
systolic pressure, mm Hg | 111.67 ± 7.67 | 163.67 ± 13.58* |
diastolic pressure, mm Hg | 74.67 ± 4.05 | 102.78 ± 9.37* |
Maternal age, years | 29.20 ± 2.73 | 29.11 ± 3.85 |
Maternal body mass index, kg/m2 | 26.13 ± 2.40 | 27.45 ± 2.70 |
Annotation: Clinical characteristics of two groups of pregnant women for immunohistochemical staining. The value was expressed as mean ± SD. The P value between the preeclampsia group and the control group was given. *P < 0.05. |
Gene set enrichment analysis (GSEA).
In order to explore the potential biological functions of hub genes in preeclampsia, GSE75010 dataset was used for GSEA analysis. The results indicated that these 5 hub genes were all enriched in the 17 pathways including T cell receptor signaling pathway, leukocyte transendothelial migration, viral myocarditis, primary immunodeficiency, FC epsilon RI signaling pathway, prion diseases, cell adhesion molecules cams, natural killer cell mediated cytotoxicity, complement and coagulation cascades, leishmania infection, systemic lupus erythematosus, antigen processing and presentation, intestinal immune network for IgA production, pantothenate and COA biosynthesis, nod like receptor signaling pathway, pathogenic Escherichia coli infection, glutathione metabolism (Table 3). These results indicated that these 5 hub genes may be involved in the development of preeclampsia via regulating these pathways.
Table 3
Pathways enriched in 5 genes in GSEA analysis
GGene Pathways | HCK | ITGAM | LCP2 | PLEK | TYROBP |
KEGG_T_CELL_RECEPTOR_SIGNALING_PATHWAY | 2.12 | 2.01 | 1.82 | 1.83 | 1.9 |
KEGG_LEUKOCYTE_TRANSENDOTHELIAL_MIGRATION | 1.99 | 2.05 | 1.81 | 1.75 | 1.97 |
KEGG_VIRAL_MYOCARDITIS | 1.88 | 1.71 | 1.62 | 1.62 | 1.72 |
KEGG_PRIMARY_IMMUNODEFICIENCY | 1.88 | 1.76 | 1.61 | 1.86 | 1.94 |
KEGG_FC_EPSILON_RI_SIGNALING_PATHWAY | 1.88 | 1.89 | 1.69 | 1.89 | 1.81 |
KEGG_PRION_DISEASES | 1.86 | 1.76 | 1.76 | 1.81 | 1.83 |
KEGG_CELL_ADHESION_MOLECULES_CAMS | 1.86 | 1.86 | 1.65 | 1.77 | 1.79 |
KEGG_NATURAL_KILLER_CELL_MEDIATED_CYTOTOXICITY | 1.84 | 1.73 | 1.52 | 1.62 | 1.8 |
KEGG_COMPLEMENT_AND_COAGULATION_CASCADES | 1.81 | 1.71 | 1.58 | 1.72 | 1.79 |
KEGG_LEISHMANIA_INFECTION | 1.8 | 1.7 | 1.64 | 1.69 | 1.67 |
KEGG_SYSTEMIC_LUPUS_ERYTHEMATOSUS | 1.77 | 1.68 | 1.65 | 1.67 | 1.62 |
KEGG_ANTIGEN_PROCESSING_AND_PRESENTATION | 1.68 | 1.66 | 1.53 | 1.57 | 1.63 |
KEGG_INTESTINAL_IMMUNE_NETWORK_FOR_IGA_PRODUCTION | 1.67 | 1.52 | 1.52 | 1.56 | 1.58 |
KEGG_PANTOTHENATE_AND_COA_BIOSYNTHESIS | 1.66 | 1.52 | 1.53 | 1.55 | 1.69 |
KEGG_NOD_LIKE_RECEPTOR_SIGNALING_PATHWAY | 1.63 | 1.65 | 1.61 | 1.74 | 1.63 |
KEGG_PATHOGENIC_ESCHERICHIA_COLI_INFECTION | 1.61 | 1.88 | 1.57 | 1.68 | 1.64 |
KEGG_GLUTATHIONE_METABOLISM | 1.59 | 1.61 | 1.53 | 1.78 | 1.53 |
Annotation: The GSE75010 was used for GSEA analysis. The values in the table represent the NES value. The table listed KEGG pathways in which all genes satisfy P < 0.05. The number in units means the NES value of each for the pathway. |