lncRNAs in Non-Malignant Tissue Have Prognostic Value in Colorectal Cancer

Abstract

Although colorectal cancer (CRC) is the third most frequent cause of cancer related death in Europe, clinically relevant biomarkers for therapy guidance and prognosis are insufficiently reliable. Long non-coding RNAs (lncRNAs) are RNAs over 200 nucleotides long that are not translated into proteins but can influence biological processes. There is emerging evidence for their involvement in solid cancer as oncogenes, tumour suppressors or regulators of cell proliferation and metastasis development. The goal of this study was to evaluate the prognostic effect of selected lncRNAs in a retrospective study on CRC patients from the Czech Republic. We used a quantitative PCR approach to measure the expression in paired non-malignant and tumour tissue samples of CRC patients of nine lncRNAs previously shown to be involved in cancer progression-ANRIL, CCAT1, GAS5, linc-ROR, MALAT1, MIR155HG, PCAT1, SPRY4-IT1 and TUG1. Associations between expression and expression ratios and clinical characteristics and survival were assessed by using univariable Cox proportional hazards models, Kaplan-Meier estimations with the Gehan-Wilcoxon test, the Mann-Whitney U test, the Kruskal-Wallis test and Spearman's correlations. A comparison of expression in tumour tissue (TT) and non-malignant mucosa tissue (MT) showed significant upregulation of CCAT1 and linc-ROR in TT (p < 0.001 and p = 0.001, respectively) and downregulation of ANRIL, MIR155HG and MALAT1 (p = 0.001, p = 0.010, p = 0.001, respectively). Linc-ROR was significantly associated with the presence of synchronous metastases (p = 0.033). For individual tissue types, lower MIR155HG expression in TT was correlated with both shorter overall survival (p = 0.008) and shorter disease-free survival (p = 0.040). In MT, expression ratios of CCAT1/ANRIL and CCAT1/MIR155HG were associated with overall survival (p = 0.005 and p = 0.006, respectively). Our results revealed that changes in expression of lncRNAs between MT and TT hold potential to be used as prognostic biomarkers in CRC patients. Moreover, the ratios of CCAT1 to ANRIL and MIR155HG in MT also exhibit potential for prognosis assessment without tumour sampling. Our results also indicate that cancer progression is associated with detrimental system-wide changes in patient tissue, which might govern patient survival even after successful elimination of tumour or cancerous cells.

Keywords: CCAT1; MIR155HG; PCAT1; colorectal carcinoma; lncRNA; lncRNA ratio.

Figure 1

Expression of nine lncRNAs (long non-coding RNAs) in tumour tissue compared to non-malignant tissue. Boxplots of nine lncRNAs showing the fold change of their expression in tumour tissue vs. non-malignant tissue. Significantly up- or downregulated lncRNAs (p < 0.05) are marked.

Figure 2

Association between MIR155HG and PCAT1 expression fold change and survival. (a) Kaplan Meier curve using optimized thresholds for fold change of MIR155HG and its association with OS (overall survival) and (b) fold change of PCAT1 and its association with DFS (disease-free survival).

Figure 3

Significant associations between lncRNAs expression fold change in tumour tissue (TT) vs. healthy tissue (MT) and tumour staging. Displayed is the ratio between healthy tissue (MT) and tumour tissue (TT). A value above 1 represents elevated expression in TT. (a) The change in expression of linc-ROR differed significantly within the categories of AJCC staging and was largest in AJCC stage 3 samples; (b) The change in expression of linc-ROR was significantly higher in patients without distant metastases; (c) For MALAT1 the fold change varied significantly between the T stages; (d) Change in ANRIL expression change was significantly higher in patients without lymph node involvement (N0).

Figure 3

Significant associations between lncRNAs expression fold change in tumour tissue (TT) vs. healthy tissue (MT) and tumour staging. Displayed is the ratio between healthy tissue (MT) and tumour tissue (TT). A value above 1 represents elevated expression in TT. (a) The change in expression of linc-ROR differed significantly within the categories of AJCC staging and was largest in AJCC stage 3 samples; (b) The change in expression of linc-ROR was significantly higher in patients without distant metastases; (c) For MALAT1 the fold change varied significantly between the T stages; (d) Change in ANRIL expression change was significantly higher in patients without lymph node involvement (N0).

Figure 4

Associations of lncRNA normalized expressions in MT and TT with overall survival. (a) Kaplan Meier curve for the relationship between CCAT1 expression in MT with OS and (b) Kaplan Meier curve for MIR155HG expression in TT and its association with OS.

Figure 5

Associations of lncRNA expressions in MT and TT with tumour characteristics. Displayed are the relative expression values in the respective tissue type, grouped by different clinical characteristics. A higher value corresponds to higher expression. (a) The expression of linc-ROR in MT differed significantly within the categories of AJCC staging; (b) The expression of MIR155HG in TT was significantly higher in patients without distant metastases; (c) linc-ROR expression in TT also significantly differed between tumour grades; (d) MALAT1 expression in TT was on the borderline of being associated with varying tumour grades.

Figure 6

Associations of expression ratios of two lncRNAs in MT with OS. (a) Table displays all possible expression ratios of the nine lncRNAs with their respective univariable Cox proportional hazard model p-values for association with OS. Each cell contains the p-value of the expression ratio of lncRNAs stated in the corresponding row and column headings. The p-values are identical for reciprocal ratios, that is, for X/Y and Y/X; (b–d) Kaplan-Meier curves with an applied optimized threshold for the relationship between OS and the expression ratio of (b) CCAT1/ANRIL in MT, (c) CCAT1/MIR155HG in MT and (d) MALAT1/ANRIL in MT.

Figure 7

Expression ratios of two lncRNAs in MT. (a) Displayed are six lncRNA expression ratios in MT that are significantly or potentially associated with OS. For the ratios featured in Figure 6 the values of the optimized threshold used for the Kaplan-Meier curves are also shown; (b) Scatter plot for the correlation between the two most prognostic ratios in MT that are associated with OS; (c) Scatter plot of the two relative expression values of lncRNAs in MT involved in the two strongest ratios except CCAT1; (d) The ratio of MALAT1/ANRIL was negatively associated with tumour stage; (e) The expression ratio CCAT1/ANRIL significantly decreased with higher AJCC stage (Spearman: p = 0.0072).