Combined identification of long non-coding RNA CCAT1 and HOTAIR in serum as an effective screening for colorectal carcinoma

Abstract

Long non-coding RNAs (lncRNAs) CCAT1 and HOTAIR have been shown to play an important regulatory role in cancer biology, and CCAT1 and HOTAIR are upregulated in several cancers, however, its value in the diagnosis of colorectal cancer (CRC) is unclear. Therefore, the aim of this study is to evaluate the clinical significance of plasma CCAT1 and HOTAIR as a biomarker in the screening of CRC. In our study, we found that the levels of HOTAIR (P < 0.05) and CCAT1 (P < 0.05) were significantly higher in plasma of CRC patients than that of the healthy control. Moreover, the levels of lincRNA-p21 (P < 0.05) were obviously decreased in plasma of CRC patients as compared to those of healthy control. There was highly correlated for CCAT1 (R = 0.752, mean differences = -0.06 ± 1.20), HOTAIR (R = 0.739, mean differences = -0.26 ± 0.76) and lincRNA-p21 (R = 0.848, mean differences = -0.41 ± 0.89) in plasma and serum. By receiver operating characteristic curve (ROC) analysis, plasma CCAT1 provided the higher diagnostic performance for detection of CRC (the area under the ROC curve (AUC), 0.836; P < 0.001; sensitivity, 75.7%; specificity, 85.3%). Moreover, CCAT1 combining with HOTAIR could provide a more effective diagnosis performance (AUC, 0.954, P < 0.001, sensitivity, 84.3%; specificity, 80.2%). Most importantly, this combination was effective to detect CRC at an early stage (85%). In conclusion, our results demonstrated that increased plasma HOTAIR and CCAT1 could be used as a predictive biomarker for CRC screening, and that combination of HOTAIR and CCAT1 had a higher positive diagnostic rate of CRC than HOTAIR or CCAT1 alone.

Keywords: CCAT1; Colorectal carcinoma; HOTAIR; long non-coding RNA; tumor biomarker.

Figure 1

Identification of tumor tissues-enriched lncRNA implicated in CRC patients. 13 CRC-related lncRNAs are filtered from lncRNA and disease database. Real-time PCR analysis is performed to determine the expression levels of lncRNAs in 32 pairs of CRC tumor tissues and corresponding non-tumourous specimens. ΔCt method is used to calculate lncRNA expression, which is normalized to GAPDH, and smaller ΔCt value indicated higher expression. Values are expressed as mean ± SD, *P < 0.05 versus non-tumourous group.

Figure 2

CCAT1, HOTAIR and lincRNA-p21 were detectable in plasma from CRC patients. CCAT1 (A), HOTAIR (B) and lincRNA-p21 (C) expression are examined by real-time PCR and normalized to GAPDH expression in plasma from CRC patients and healthy controls. Circulating lncRNAs expressions are calculated using ΔCt method. Values are expressed as mean ± SD, n = 32 in each group, *P < 0.05 versus healthy controls.

Figure 3

Correlation of lncRNAs levels between plasma and serum in CRC patients. Linear correlation plot of CCAT1 (A), HOTAIR (B) and lincRNA-p21 (C) in plasma and serum (left). There is a high correlation comparing the indicated lncRNAs levels between plasma and serum. Bland-Altman plot of the difference between plasma and serum CRC-related lncRNAs level versus their average. Horizontal solid lines in the middle represent the mean difference. Upper and lower solid lines represent the limits of agreement (95% confidence intervals) (right).

Figure 4

HOTAIR, CCAT1 and lincRNA-p21 levels in pre-operative and post-operative plasma samples. CRC-related lncRNAs CCAT1 (A), HOTAIR (B) and lincRNA-p21 (C) levels are examined by real-time PCR in post-operative samples as compared to pre-operative samples. Circulating lncRNAs expressions are calculated using ΔCt method. Values are expressed as mean ± SD, n = 32 in each group.

Figure 5

Evaluation of HOTAIR, CCAT1 or lincRNA-p21 in plasma as a predictive CRC-related biomarker. Receiver operating characteristics (ROC) curves are drawn with the data of plasma lncRNAs, CCAT1 (A), HOTAIR (B) and lincRNA-p21 (C), from 32 CRC patients and 32 healthy controls. ROC corves of a combination of HOTAIR and CCAT1 to discriminate CRC from healthy controls (D).