Lowly expressed ribosomal protein s19 in the feces of patients with colorectal cancer

Abstract

Colorectal cancer (CRC) has become one of the most common fatal cancers. CRC tumorigenesis is a complex process involving multiple genetic changes to several sequential mutations or molecular alterations. P53 is one of the most significant genes; its mutations account for more than half of all CRC. Therefore, understanding the cellular genes that are directly or indirectly related to p53 is particularly crucial for investigating CRC tumorigenesis. In this study, a p53-related ribosomal protein, ribosomal protein S19 (RPS19), obtained from the feces of CRC patients is evaluated by using specifically quantitative real-time PCR and knocked down in the colonic cell line by gene silencing. This study found that CRC patients with higher expressions of RPS19 in their feces had a better prognosis and consistent expressions of RPS19 and BAX in their colonic cells. In conclusion, the potential mechanism of RPS19 in CRC possibly involves cellular apoptosis through the BAX/p53 pathway, and the levels of fecal RPS19 may function as a prognostic predictor for CRC patients.

Figure 1

Six differentially expressed p53-associated ribosomal proteins in feces of CRC patients. Differentially expressed genes with statistic significance (P < 0.05) are grouped by average-linkage hierarchical clustering. Each row represents a gene and each column is a sample. Group L, five patients (one at Dukes' stage B, one at stage C, and three at stage D). Group E, six patients (two at Dukes' stage A, two at stage B, and two at stage C) and two normal control pools (NP01 and NP02). NP01, pooled by two healthy men; NP02, pooled by three healthy women. A region cluster depicts the genes based on the similarity between their expressions in cases. High expression is shown in deep yellow, low expression in blue. Arrow indicates RPS19.

Figure 2

Receiver operating characteristic (ROC) curve for fecal RPS19. The points on the curve represent the relative mRNA levels of RPS19 in the feces and the sensitivity and (1-specificity) of the marker for overall survival.

Figure 3

The Kaplan-Meier overall survival curves in patients with colorectal cancer according to fecal RPS19. The relative mRNA levels of RPS19 in the feces are stratified into two groups: RPS19⁻ (<2.76 × 10⁻⁵) and RPS19⁺ (≥2.76 × 10⁻⁵). The six-year overall survival rate of the RPS19⁺ group (n = 62) is better than that of the RPS19⁻ group (P = 0.008, log-rank test).

Figure 4

Efficiency of RPS19 silence in colonic cells by RNA interference. RPS19 silence is achieved by the lentivirus-mediated RNAi experiment. Relative mRNA levels of RPS19 are quantified by qRT-PCR with TaqMan probes and normalized by individual level of 18 s rRNA. The relative expression level of shLuc-infected cells is considered as 1. Results are representative of those obtained in two-to-three separate experiments with error bars showing standard error. Changes of protein levels are immunoblotted with antibodies against RPS19 and β-actin (in black square).

Figure 5

Changes of BAX expression in RPS19-silent colonic cells. Relative mRNA levels of BAX are quantified by qRT-PCR with TaqMan probes and normalized by individual level of 18 s rRNA. The relative expression level of shLuc-infected cells is considered as 1. Results are representative of those obtained in one experiment.