Development and Validation of a Prognostic Model of RNA-Binding Proteins in Colon Adenocarcinoma: A Study Based on TCGA and GEO Databases

Figures & data

Table 1 Primer Sequences Used in the qRT-PCR Assay

Primer	Sequence (5′–3′)
RPL10L-For	AGGGTTCACATTGGCCAAGTT
RPL10L-Rev	TAAGAGGGGGGCAGCACA
ERI1-For	AGTGTCAACTCAGCAGGCTC
ERI1-Rev	TGAGGCCGCCCATCATAATC
POP1-For	ATCGACTAGGCTGGGGAAGT
POP1-Rev	AGTCTGGAAAATCGCCTGGG
CAPRIN2-For	AGCAATGGTAGCCTTGCCTT
CAPRIN2-Rev	TCCCACCACGAGTACATCCT
TDRD7-For	TGGCCTCATATTTGCACGGT
TDRD7-Rev	ACCCTTTCAACGGTGGTGTT
SNIP1-For	GCTTTGTGGACCAGGTGTTT
SNIP1-Rev	TGTACAGTCACGGGCTTGAG
PPARGC1-For	CACGGACAGAACTGAGGGAC
PPARGC1-Rev	TTCGTTTGACCTGCGCAAAG
GAPDH-For	CTTTGGTATCGTGGAAGGA
GAPDH-Rev	CACCCTGTTGCTGTAGCC

Figure 1 Identification of differentially expressed RNA-binding proteins (DE RBPs) in The Cancer Genome Atlas (TCGA) dataset. (A) Heat map of the DE RBPs based on their log2-transformed fragments per kilobase of transcript per million mapped reads (FPKM) expression values. Red represents high expression, and green represents low expression. (B) Volcano plot of DE RBPs between colon adenocarcinoma (COAD) and normal colon tissue; 208 were upregulated, and 122 were downregulated. Red: upregulated RBPs; black: unchanged RBPs; green: downregulated RBPs.

Figure 2 Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of differentially expressed RBPs (DE RBPs). (A) Bubble plot of enriched GO terms. (B) Bubble plot of enriched KEGG pathways.

Figure 3 Protein-protein interaction (PPI) network and modules analysis. (A) PPI network for differentially expressed RBPs (DE RBPs). (B) Key module 1 in the PPI network.

Figure 4 Selection of prognosis-related RBPs in the training cohort. (A) Univariate Cox regression analysis. (B and C) Least absolute shrinkage and selection operator (LASSO) regression analysis. (D) Multivariate Cox regression analysis to screen out the key RNA-binding proteins (RBPs) most relevant to prognosis.

Table 2 Seven Prognosis-Associated Hub RNA-Binding Proteins (RBPs) Identified by Multivariate Cox Regression Analysis

RBP Name	Coef	HR	Lower 95% CI	Upper 95% CI	P-value
RPL10L	−0.066	0.936	0.871	1.006	0.071
ERI1	−0.398	0.672	0.494	0.913	0.011
POP1	−0.527	0.591	0.401	0.869	0.008
CAPRIN2	0.247	1.280	0.985	1.664	0.065
TDRD7	−0.364	0.695	0.443	1.090	0.113
SNIP1	−0.442	0.643	0.353	1.168	0.147
PPARGC1A	−0.199	0.820	0.733	0.916	<0.001

Table 3 The Prognostic Value of Different Clinical Parameters in the Cancer Genome Atlas (TCGA) Cohort

	Univariate Analysis			Multivariate Analysis
	HR	95% CI	P-value	HR	95% CI	P-value
Age	1.030	1.011–1.049	0.002	1.049	1.028–1.070	<0.001
Gender	1.157	0.763–1.756	0.493	0.855	0.555–1.317	0.478
Stage	2.120	1.665–2.699	<0.001	2.094	1.632–2.687	<0.001
Risk score	1.417	1.287–1.559	<0.001	1.441	1.278–1.625	<0.001

Figure 5 Risk score analysis of the seven-gene prognostic model in The Cancer Genome Atlas (TCGA) cohort. (A) Survival curve for low-risk and high-risk groups. (B) Receiver operating characteristic (ROC) curves for predicting overall survival (OS) based on the risk score. (C) Expression heat map, risk score distribution, and survival status.

Figure 6 Risk score analysis of the eight-gene prognostic model in the GEO database combined cohort. (A) Survival curve for low-risk and high-risk groups. (B) Receiver operating characteristic (ROC) curves for predicting overall survival (OS) based on the risk score. (C) Expression heat map, risk score distribution, and survival status.

Table 4 The Prognostic Value of Different Clinical Parameters in the GEO Database Combined Cohort

	Univariate Analysis			Multivariate Analysis
	HR	95% CI	P-value	HR	95% CI		P-value
Age	1.021	1.012–1.031	<0.001	1.026	1.017–1.035		<0.001
Gender	1.122	0.897–1.402	0.313	1.116	0.891–1.398		0.340
Stage	2.274	1.966–2.631	<0.001	2.300	1.978–2.674		<0.001
Risk score	2.074	1.640–2.623	<0.001	1.624	1.254–2.101		<0.001

Figure 7 Nomogram (A) for predicting the 1-, 3-, and 5-year overall survival (OS) of colon adenocarcinoma (COAD) patients in The Cancer Genome Atlas (TCGA) cohort. (B) Calibration curves for Nomogram.

Figure 8 Verification of hub RNA-binding protein (RBP) expression in colon adenocarcinoma (COAD) tissues and normal colon tissues using the Human Protein Atlas (HPA) database. (A) CAPRIN2, (B) ERI1, (C) RPL10L, (D) SNIP1 and (E) TDRD7.

Figure 9 Kaplan-Meier curve analysis of the three prognostic RNA-binding proteins (RBPs), including CAPRIN2, POP1 and PPARGC1A, in The Cancer Genome Atlas (TCGA) cohort.

Figure 10 qRT-PCR validation of seven prognosis-related RBP-encoding genes in colon adenocarcinoma (COAD) cell models compared to control cells. *p < 0.05. **p < 0.01. ***p < 0.001.

Abbreviation: ns, not significant.