CSTF2 Acts as a Prognostic Marker Correlated with Immune Infiltration in Hepatocellular Carcinoma

Figures & data

Figure 1 The transcriptional level of CSTF2 in HCC. (A) CSTF2 transcriptional levels in various cancers analyzed in the TIMER database. (B–F) The expression of CSTF2 in HCC based on TCGA database (B), GSE45436 (C), GSE84402 (D), GSE105130 (E), and ICGC database (F). (G) Elevated mRNA levels of CSTF2 in the hepatocellular carcinoma cell line HepG2.

Note: * p<0.05, ** p<0.01, *** p<0.001.

Figure 2 CSTF2 protein was upregulated in HCC. (A) The protein expression of CSTF2 in HCC based on the HPA database. (B) The protein expression of CSTF2 in cell lines.

Table 1 Relationship Between CSTF2 and Clinicopathological Characteristics of HCC Patients

Classification	Total	CSTF2 Expression		χ^2	P value
		Low (n=172)	High (n=172)
Age
≤60	171	88	83
>60	173	84	89	0.291	0.590
Gender
Male	234	108	126
Female	110	64	46	4.330	0.037
AJCC Stage
I	170	99	71
II–IV	174	73	101	9.118	0.003
Histologic grade
G1	46	28	18
G2	168	88	80
G3	118	49	69
G4	12	7	5	6.278	0.099
Status
Alive	233	128	105
Dead	111	44	67	7.036	0.008

Notes: AJCC stage, according to the 7th edition AJCC staging system.

Table 2 Univariate and Multivariate Cox Regression Analyses Were Conducted to Identify Independent Risk Factors for Poor Survival of HCC

Variables	Univariate Analysis			Multivariate Analysis
	HR	95% CI	P value	HR	95% CI	P value
Age	1.021	1.001–1.040	0.035	1.022	1.002–1.042	0.027
AJCC stage	1.475	1.148–1.895	0.002	1.405	1.083–1.824	0.010
CSTF2	1.703	1.202–2.412	0.003	1.659	1.147–2.399	0.007
Gender	0.630	0.396–1.001	0.051	–	–	–
Histologic grade	1.224	0.893–1.678	0.210	–	–	–
Family history	1.486	0.943–2.343	0.088	–	–	–
Vascular invasion	1.466	0.911–2.360	0.115	–	–	–

Notes: AJCC stage, according to the 7th edition AJCC staging system.

Figure 3 Diagnostic and prognostic values of CSTF2 in HCC based on TCGA database. (A) The diagnostic capability of CSTF2 was evaluated in the TCGA-LIHC cohort. (B) The relationship of CSTF2 expression and overall survival was evaluated in GEPIA with TCGA data.

Figure 4 Establishment and validation of a prediction nomogram in TCGA and ICGC cohorts. (A) The CSTF2-based nomogram for predicting 1-, 3-, and 5-year overall survival rates of HCC patients in the TCGA-LIHC cohort. (B) Calibration curves of 1-, 3-, and 5-year overall survival for HCC patients in the TCGA-LIHC cohort. (C) Calibration curves of 1- and 3-year overall survival for HCC patients in the ICGC cohort.

Abbreviation: OS, overall survival.

Figure 5 Diagnostic and prognostic values of CSTF2 in HCC based on the ICGC database. (A) The diagnostic capability of CSTF2 was validated in the ICGC cohort. (B) The relationship of CSTF2 expression and overall survival was validated in the ICGC cohort. (C and D) The independent risk factors were identified in the ICGC cohort by univariate (C) and multivariate (D) Cox regression analyses.

Figure 6 Functional enrichment analyses of CSTF2-related genes in HCC. (A) The top 50 positively correlated genes of CSTF2 are displayed by a heatmap. (B) The top 50 negatively correlated genes of CSTF2 are displayed by a heatmap. (C) GO enrichment analysis of the top 50 positively correlated genes of CSTF2. (D) KEGG enrichment analysis of the top 50 positively correlated genes of CSTF2.

Abbreviations: GO, gene ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; BP, biological process; CC, cellular component; MF, molecular function.

Table 3 Correlation Analysis of CSTF2 with Immune Cell Markers in the TIMER Database

Terms	Gene Marker	None		Purity
		Cor	P value	Cor	P value
T cell exhaustion	PDCD1(PD-1)	0.153	***	0.187	***
	CTLA4	0.163	***	0.219	***
	LAG3	0.115	*	0.138	*
	HAVCR2(TIM3)	0.297	***	0.391	***
	GZMB	0.065	0.212	0.084	0.120
	BTLA	0.149	**	0.197	***
	CD244(SLAMF4)	0.130	*	0.166	**
	CD274(PD-L1)	0.442	***	0.475	***
	CD96	0.145	**	0.213	***
	IDO1	0.218	***	0.248	***
	KDR	0.207	***	0.240	***
	PDCD1LG2(PD-L2)	0.233	***	0.305	***
	TGFBR1	0.492	***	0.480	***
	TIGIT	0.206	***	0.285	***
T cell (general)	CD3E	0.073	0.160	0.135	*
	CD3G	0.220	***	0.274	***
	CD28	0.352	***	0.412	***
	CD2	0.061	0.239	0.122	*
CD8+ T cell	CD8A	0.124	*	0.166	**
	CD8B	0.078	0.136	0.118	*
CD4+ T cell	CD4	0.209	***	0.253	***
	CD40LG(CD40L)	0.067	0.201	0.136	*
	CXCR4	0.276	***	0.352	***
Th1	TBX21	0.084	0.105	0.140	**
	STAT4	0.079	0.129	0.112	*
	STAT1	0.407	*	0.428	***
	IFNG	0.180	***	0.222	***
	TNF	0.265	***	0.349	***
Th2	STAT6	0.374	***	0.359	***
	STAT5A	0.399	***	0.424	***
	GATA3	0.121	*	0.196	***
	CCR8	0.381	***	0.447	***
Tfh	BCL6	0.368	***	0.365	***
	IL-21	0.111	*	0.149	**
Th17	STAT3	0.398	***	0.416	***
	IL17A	0.042	0.423	0.071	0.190
Treg	FOXP3	0.251	***	0.299	***
	STAT5B	0.487	*	0.474	***
	TGFB1	0.223	***	0.28	***
	IL2RA(CD25)	0.293	***	0.365	***
B cell	CD19	0.167	**	0.192	***
	CD79A	0.039	0.451	0.086	0.111
Monocyte	CD86(B7-2)	0.316	***	0.411	***
	CSF1R	0.284	***	0.376	***
TAM	CCL2	0.150	**	0.203	***
	CD68	0.301	***	0.357	***
	IL10	0.259	***	0.321	***
M1 macrophage	IRF5	0.383	***	0.378	***
	PTGS2	0.173	***	0.247	***
	NOS2	0.154	**	0.164	**
	ARG2	0.270	***	0.286	***
M2 macrophage	CD163	0.278	***	0.357	***
	VSIG4	0.303	***	0.377	***
	MS4A4A	0.268	***	0.362	***
	MRC1	0.146	**	0.181	***
Neutrophils	CEACAM8	0.060	0.248	0.076	0.160
	ITGAM	0.405	*	0.458	***
	CCR7	0.069	0.185	0.132	*
	MPO	0.133	*	0.154	**
	CD32(FCGR2A)	0.467	*	0.510	***
Natural killer cell	FCGR3A(CD16)	0.383	***	0.444	***
	NCAM1(CD56)	0.231	***	0.262	***
	KIR2DL1	0.023	0.658	−0.001	0.991
	KIR2DL3	0.162	**	0.194	***
	KIR2DL4	0.199	***	0.220	***
	KIR3DL1	0.071	0.172	0.088	0.101
	KIR3DL2	0.083	0.110	0.115	*
	KIR2DS4	0.067	0.197	0.082	0.130
Dendritic cell	HLA-DRA	0.274	***	0.348	***
	HLA-DPA1	0.240	***	0.313	***
	CD1C	0.089	0.0854	0.137	*
	NRP1	0.429	*	0.443	***
	ITGAX	0.296	***	0.380	***
	THBD	0.080	0.123	0.122	*

Notes: Purity, correlation adjusted by purity. *P<0.05, **P<0.01, ***P<0.001.

Abbreviations: Th, T helper cell; Tfh, follicular helper T cell; Treg, regulatory T cell; TAM, tumor-associated macrophage.

Figure 7 Relationship between CSTF2 expression and immune cell infiltration. (A) The distribution of 22 immune cell subpopulations between the CSTF2 high (red) and low (blue) expression groups. (B) The correlation of CSTF2 expression with immune cells was analyzed in the TIMER database. (C–G) CSTF2 expression was positively correlated with the immune checkpoints PD-L1 (C), CTLA4 (D), TIM3 (E), PD-1 (F) and TIGIT (G). (H) The expression of immune checkpoints between the CSTF2 high (red) and low (blue) expression groups.

Note: *p<0.05, **p<0.01, ***p<0.001, ns, not significant.

Figure 8 CSTF2 silencing alleviated the malignant phenotypes in hepatic cancer cells. (A) The mRNA expression of CSTF2 in HepG2 cells was significantly inhibited by si-CSTF2#3 transfection. (B) CSTF2 protein in HepG2 cells was significantly inhibited by si-CSTF2#3 transfection. (C) CSTF2 silencing inhibited the proliferation of HepG2 cells. (D) The protein expression of Bcl-2 (anti-apoptotic protein) and Bax (pro-apoptotic protein) in transfected HepG2 cells. (E) CSTF2 silencing increased the apoptosis of HepG2 cells. (F) CSTF2 silencing reduced the migration and invasion of HepG2 cells.

Note: ***p<0.001.

Figure 9 CSTF2 silencing inactivated the Wnt/β-catenin signaling pathway. (A) GSEA revealed that the Wnt/β-catenin signaling pathway was enriched in CSTF2 high expression group based on TCGA-LIHC cohort. (B) CSTF2 and Wnt/β-catenin signaling pathway-related protein expression in transfected HepG2 cells.