A long non-coding RNA signature to improve prognosis prediction of colorectal cancer

Abstract

Increasing evidence suggests long non-coding RNAs (lncRNAs) are frequently aberrantly expressed in cancers, however, few related lncRNA signatures have been established for prediction of cancer prognosis. We aimed to develop a lncRNA signature to improve prognosis prediction of colorectal cancer (CRC). Using a lncRNA-mining approach, we performed lncRNA expression profiling in large CRC cohorts from Gene Expression Ominus (GEO), including GSE39582 test series(N=436), internal validation series (N=117); and two independent validation series GSE14333 (N=197) and GSE17536(N=145). We established a set of six lncRNAs that were significantly correlated with the disease free survival (DFS) in the test series. Based on this six-lncRNA signature, the test series patients could be classified into high-risk and low-risk subgroups with significantly different DFS (HR=2.670; P<0.0001). The prognostic value of this six-lncRNA signature was confirmed in the internal validation series and another two independent CRC sets. Gene set enrichment analysis (GSEA) analysis suggested that risk score positively correlated with several cancer metastasis related pathways. Functional experiments demonstrated three dysregulated lncRNAs, AK123657, BX648207 and BX649059 were required for efficient invasion and proliferation suppression in CRC cell lines. Our results might provide an efficient classification tool for clinical prognosis evaluation of CRC.

Figure 1. Kaplan–Meier estimates of the disease free survival (DFS) or disease specific survival (DSS) of GEO patients using the six-lncRNA signature

The Kaplan–Meier plots were used to visualize the DFS probabilities for the low-risk versus high-risk group of patients based on the median risk score from the GSE39582 test series patients. (A) Kaplan Meier curves for GSE39582 test series patients (N=436); (B) Kaplan–Meier curves for GSE39582 validation series patients (N=117); (C) Kaplan–Meier curves for the entire GSE39582 patients (combined test and validation series patients, N=553). (D) Kaplan– Meier curves for GSE14333 patients (N=197); Kaplan–Meier curves for (E) DFS and (F) DSS of GSE17536 patients (N=145); The tick marks on the Kaplan–Meier curves represent the censored subjects. The differences between the two curves were determined by the two-sided log-rank test. The number of patients at risk was listed below the survival curves.

Figure 2. LncRNA risk score analysis of entire GSE39582 series

The distribution of six-lncRNA risk score, patients' survival status and lncRNA expression signature were analyzed in the entire GSE39582 series patients (N=553). (A) LncRNA risk score distribution; (B) patients' survival status and time; (C) heatmap of the lncRNA expression profiles. Rows represent lncRNAs, and columns represent patients. The black dotted line represents the median lncRNA risk score cutoff dividing patients into low-risk and high-risk groups.

Figure 3. Kaplan–Meier estimates of the DFS of GSE39582 and GSE17536 patients with known AJCC stage (N=698)

Patients were first stratified on the basis of AJCC stage (stage I and II vs stage III and IV) of their tumor samples. Kaplan–Meier plots were then used to visualize the survival probabilities for the low-risk versus high-risk group of patients based on the median risk score from the dataset patients within each stratum. (A) Kaplan–Meier curves for all patients (N=698); (B) Kaplan–Meier curves for stage I and II patients (N=371); (C) Kaplan–Meier curves for stage III and IV patients (N=327). The number of patients at risk was listed below the survival curves. Kaplan–Meier estimates of the DFS of GSE39582 and GSE14333 patients with known chemotherapy status. (D) Kaplan–Meier curves for all patients (N=622); (E) Kaplan–Meier curves for patients without chemotherapy (N=355); (F) Kaplan–Meier curves for patients with chemotherapy (N=267). The tick marks on the Kaplan–Meier curves represent the censored subjects. The differences between the two curves were determined by the two-sided log-rank test.

Figure 4. (A) Gene Set Enrichment Analysis Delineates Biological Pathways and Processes associated with risk score

Cytoscape and Enrichment Map were used for visualization of the GSEA results. Nodes represent enriched gene sets, which are grouped and annotated by their similarity according to related gene sets. Enrichment results were mapped as a network of gene sets (nodes). Node size is proportional to the total number of genes within each gene set. Proportion of shared genes between gene sets is represented as the thickness of the green line between nodes. (B) Risk score of patients with or without distant metastasis in combined GSE39582 and GSE17536 data (N=698, P=0.0001).

Figure 5. Box plot of (A) AK024680, (B) CR622106 and (C) AK026784 and expression in CRC with genomic amplification (Amp; N=76, N=73 and N=237, respectively) and without genomic amplification (W/o amp; N=326, N=329 and N=165, respectively)

MWU test was used to determine the significance of the comparisons. Expression of (D) AK123657, (E) BX648207 and (F) BX649059 were quantified by qRT-PCR in 25 pairs of CRC tissues and their matched normal tissues. AK123657 (P=0.0007, Paired ttest), BX648207 (P=0.0188, Paired ttest) and BX649059 (P<0.0001, Paired ttest) expression were significantly decreased in CRC tissues compared with their normal tissues. To evaluate the effects of AK123657, BX648207 and BX649059 on cell biological behaviors, specific small interfering RNAs (siRNAs) were employed to knockdown the lncRNAs expression in human CRC HCT116 (G) and SW1116 (H) cells. Cell-counting kit-8 assays indicated that cell proliferation was increased when AK123657, BX648207 and BX649059 were knockdown (I & J). We further used Transwell assay to monitor the effect of manipulating AK123657, BX648207 and BX649059 expression on cell invasiveness. Knockdown of AK123657, BX648207 and BX649059 significantly increase the number of HCT116 and SW1116 cells that penetrated the Transwell filter (K), which suggested a substantial gain of cell invasion ability.