Identifying the Prognostic Risk Factors of Synaptojanin 2 and Its Underlying Perturbations Pathways in Hepatocellular Carcinoma

Figures & data

Figure 1. SYNJ2 levels in different tissues and cell lines. (a) SYNJ2 levels in various cancer and non-cancerous tissues. The expression of liver HCC and non-HCC tissues were marked in the red box. The red dots and green dots, respectively, represent the expression of HCC and non-HCC samples. (b) Expression of SYNJ2 in liver-related tumorous cell lines. The box plot was marked in the red box

Table 1. Clinical and pathological parameters of SYNJ2 in HCCs

Variables	Terms	N	Mean ± SD	t	P-value
Tissue	HCC	371	9.7224 ± 1.1839	6.2447	1.368E-08
	Non-HCC	50	8.9827 ± 0.7161
Gender	Male	250	9.5726 ± 1.2235	−3.5583	4.220E-04
	Female	121	10.0320 ± 1.0356
Age	≥60	201	9.6082 ± 1.1112	1.9967	0.0466
	<60	169	9.8541 ± 1.2574
Grade	G1-G2	232	9.6053 ± 1.0898	−2.6794	0.0077
	G3-G4	134	9.9472 ± 1.3124
Stage	I–II	257	9.6183 ± 1.1747	−1.9216	0.0554
	III–IV	90	9.8987 ± 1.1884
Pathologic T	T1-T2	275	9.6574 ± 1.1664	−2.0143	0.0447
	T3-T4	93	9.9421 ± 1.2139
Pathologic N	N0	252	9.7033 ± 1.1966	−1.2379	0.2169
	N1	4	10.4466 ± 0.6153
Pathologic M	M0	266	9.6866 ± 1.1865	0.3093	0.7573
	M1	4	9.5014 ± 1.3792

HCC: hepatocellular carcinoma. SD: standard deviation.

Figure 2. Levels and efficacy of SYNJ2 in clinical parameters. (a) Scatter plots and ROC curve of SYNJ2 in HCC and non-HCC tissues. (b) Scatter plots and ROC curve of SYNJ2 in male and female HCC patients. (c) Scatter plots and ROC curve of SYNJ2 in young (<60) and elder (≥60) HCC cases. (d) Scatter plots and ROC curve of SYNJ2 in G1-G2 and G3-G4 HCCs. (e) Scatter plots and ROC curve of SYNJ2 in T1-T2 and T3-T4 HCCs. (f) Overall survival status of HCCs with different SYNJ2 levels

Figure 3. Visualization of multivariate Cox regression analyses and prognostic nomogram predicting. (a) Forest plot of hazard ratio showing the estimation of SYNJ2 expression and various clinicopathological parameters in HCC. (b) Nomogram predicting overall survival for significant clinicopathological parameters of HCC patients, including SYNJ2 level, gender, age, grade and T stage. (c, d) Calibration plots of the nomogram predictive performance of 1- and 5-year overall survival based on five variables (SYNJ2 level, gender, age, grade and T stage). The y-axis and x-axis separately represent actual and nomogram-predicted overall survival rate. The 45° gray line represents an ideal prediction and the black line means the nomogram-predicted performance

Table 2. Characteristics of the merged datasets

	Dataset	HCC			Non-HCC
ID	N	N	Mean	SD	N	Mean	SD
Affymetrix	28	1598	4.4432	0.3196	1316	4.2891	0.2163
Illumina	24	1061	3.7715	0.4654	770	3.6347	0.3562
Agilent	7	176	2.2357	0.5557	121	1.9751	0.5047
Rossetta	2	368	3.4306	2.9036	386	1.5999	3.3328
Human-6k	1	80	11.8181	0.7699	307	10.3700	0.9203
HiSeq × Ten	1	35	2.1138	0.5467	35	1.1540	0.3877
ArrayStar	1	26	7.8364	1.0932	30	7.3109	0.5077
GeneChip	1	5	4.5455	0.1995	5	4.4678	0.0776
NimbleGen	1	8	9.0550	0.3358	8	8.9982	0.2747
TCGA-GTEx	2	371	9.5031	1.1708	225	9.4723	0.7510

HCC: hepatocellular carcinoma. SD: standard deviation.

Figure 4. Levels and ROC diagrams of merged SYNJ2 expression data. Scatter plots and ROCs of merged SYNJ2 expression in (a) Affymetrix platform, (b) Illumina platform, (c) Agilent platform, (d) Rosetta platform, (e) Human-6k platform, (f) HiSeq×Ten platform, (g) ArrayStar platform, (h) GeneChip platform, (i) NimbleGen platform, and (j) TCGA-GTEx platforms

Figure 5. Results of SYNJ2 levels and diagnostic value. (a) Forest plots for SYNJ2 levels in HCC based on in-house IHC and different platforms. (b) SROC curve demonstrating performance of SYNJ2 in diagnosing HCC. (c) Pooled sensitivity and (d) specificity forest plots of SYNJ2

Figure 6. Alterations and overall survival status of SYNJ2 in HCC. (a) Diagnostic age, gender, and SYNJ2 altered types of HCC patients. (b) Difference of overall survival rates between SYNJ2 altered and unaltered groups

Table 3. Top 10 significant Kyoto Encyclopedia of Genes and Genome pathways of SYNJ2 and differentially coexpressed genes

Term	Count	Genes	Fold Enrichment	P-value	FDR
Metabolic pathways	59	HEXA, HEXB, IL4I1, GLDC, ASPA, TDO2, SCLY, MAT1A, CYP7A1, HDC, GSTZ1, SYNJ2, HPD, AADAT, HMGCLL1, CYP1A1, ACADS, HAL, ALDH3B2, CYP26A1, ACADL, TAT, GLUL, CTH, SDS, AKR1B10, HAO2, UROC1, AKR1D1, OAT, PRODH, BCAT1, FUT5, AASS, KMO, GLS2, ARG1, MTHFS, CSAD, ADH4, HK3, ALDH4A1, ENO3, CDA, FUT2, INPP5B, BCKDHA, NOS1, AMACR, FTCD, ACACB, DBH, PCK1, CEL, AMDHD1, GCK, AGXT2, CYP4F2, CBS	4.8963	6.60E-35	7.41E-32
Biosynthesis of antibiotics	17	AADAT, BCAT1, BCKDHA, AASS, TAT, PCK1, GLDC, ARG1, CTH, GCK, SDS, HK3, HAO2, ENO3, OAT, CBS, PRODH	8.1120	1.05E-10	1.19E-07
Histidine metabolism	7	AMDHD1, ASPA, HDC, FTCD, HAL, ALDH3B2, UROC1	32.1878	4.49E-08	5.04E-05
Biosynthesis of amino acids	9	BCAT1, ARG1, GLUL, CTH, MAT1A, SDS, ENO3, TAT, CBS	12.6452	3.86E-07	4.34E-04
Tyrosine metabolism	7	ADH4, GSTZ1, ALDH3B2, IL4I1, DBH, TAT, HPD	20.2324	8.84E-07	9.94E-04
Primary bile acid biosynthesis	5	CYP39A1, CYP46A1, CYP7A1, AMACR, AKR1D1	29.7535	1.78E-05	0.0200
Alanine, aspartate and glutamate metabolism	6	GLS2, GLUL, ASPA, ALDH4A1, IL4I1, AGXT2	17.3420	1.95E-05	0.0219
Cysteine and methionine metabolism	6	CTH, MAT1A, SDS, IL4I1, TAT, CBS	15.9729	2.95E-05	0.0332
Glycolysis/Gluconeogenesis	6	GCK, ADH4, HK3, ENO3, ALDH3B2, PCK1	9.0593	4.56E-04	0.5119
Glycine, serine and threonine metabolism	5	CTH, SDS, AGXT2, CBS, GLDC	12.9695	5.23E-04	0.5865

FDR: false discovery rate.

Figure 7. Enrichment analysis of differentially coexpressed genes of SYNJ2. (a) Venn diagram of coexpressed genes and differentially expressed genes. (b) Bar plots of biological process, (c) cell component, and (d) molecular function. (e) KEGG pathways of co-functioned SYNJ2 and differentially coexpressed genes. X axis represents the counts of involved genes and Y axis means enriched annotations and pathways

Figure 8. Diverse interactions of proteins. (a) Protein–protein interaction networks of metabolism-related differentially coexpressed genes. Each node means a gene and the bigger node size represents the higher interaction degree. The brighter node color represents the higher clustering coefficient. (b) Highly interconnected protein complexes

Figure 9. Correlations of metabolism pathways related differentially coexpressed genes based on CCLE

Table 4. Validated TF-target relationships of SYNJ2 and INPP5B in liver

Target	No. of datasets	Transcription factor	The peak close to transcription starting site
			Chromesome	Peak start	Peak end	Enrichment score for the peak
SYNJ2	6	CTCF	Chr19	45,079,122	45,079,256	4.63
	4	CREB1	Chr19	45,079,135	45,079,311	30
	4	GABPA	Chr19	45,079,129	45,079,489	141
	3	POLR2A	Chr19	45,079,053	45,079,364	9.69
INPP5B	8	CTCF	Chr1	37,940,513	37,940,620	5.23
	5	CREB1	Chr1	37,946,977	37,947,186	6.11
	4	GABPA	Chr1	37,947,007	37,947,156	3.74
	4	POLR2A	Chr1	37,946,764	37,947,036	5.09
	3	HNF4A	Chr1	37,947,028	37,947,145	4.23

TF: transcription factor.

Table 5. Genetic diseases related to transcription factors of SYNJ2 and INPP5B

	Database
Transcription factor	GAD	FunDO	NHGRI GWAS Catalog	KEGG DISEASE	OMIM
CTCF	Metabolic, HDL cholesterol, HDL lipoproteins	Lung cancer, Nephroblastoma, Breast cancer	HDL cholesterol	Mental and behavioral disorders, Autosomal dominant mental retardation	Mental retardation, autosomal dominant 21
CREB1	Pharmacogenomic, Suicide	-	-	-	Histiocytoma, angiomatoid fibrous, somatic
POLR2A	Metabolic, Triglycerides, Sex hormone-binding globulin	Adenovirus infection	-	-	-
GABPA	-	-	-	-	-

Figure 10. Representative images of SYNJ2 protein expression. Negative SYNJ2 IHC staining in normal liver (a, b) and tumor adjacent liver tissues (c, d). Positive diffused staining of SYNJ2 in HCC cells (e, f). The original images were magnified 400 times

Figure 11. Expression of SYNJ2 in normal liver and HCC from the Human Protein Atlas database. (a, b) Low diffused staining of SYNJ2 in cytoplasmic and membranous of hepatocytes from samples 3402 and 2429. (c, d) Diffusedly brown medium staining of SYNJ2 in HCC tissues from patient 3196. The original images were magnified 400 times

Data availability

The data used to support the findings during the current study are available from the corresponding author upon reasonable request.