The combination of circulating long noncoding RNAs AK001058, INHBA-AS1, MIR4435-2HG, and CEBPA-AS1 fragments in plasma serve as diagnostic markers for gastric cancer

Abstract

Background: Suitable diagnostic markers for cancers are urgently required in clinical practice. Long non-coding RNAs, which have been reported in many cancer types, are a potential new class of biomarkers for tumor diagnosis.

Results: Five lncRNAs, including AK001058, INHBA-AS1, MIR4435-2HG, UCA1 and CEBPA-AS1 were validated to be increased in gastric cancer tissues. Furthermore, we found that plasma level of these five lncRNAs were significantly higher in gastric cancer patients compared with normal controls. By receiver operating characteristic analysis, we found that the combination of plasma lncRNAs with the area under the curve up to 0.921, including AK001058, INHBA-AS1, MIR4435-2HG, and CEBPA-AS1, is a better indicator of gastric cancer than their individual levels or other lncRNA combinations. Simultaneously, we found that the expression levels of a series of MIR4435-2HG fragments are different in gastric cancer plasma samples, but most of them higher than that in healthy control plasma samples.

Materials and methods: LncRNA gene expression profiles were analyzed in two pairs of human gastric cancer and adjacent non-tumor tissues by microarray analysis. Nine gastric cancer-associated lncRNAs were selected and assessed by quantitative real-time polymerase chain reaction in gastric tissues, and 5 of them were further analyzed in gastric cancer patients' plasma.

Conclusions: Our results demonstrate that certain lncRNAs, such as AK001058, INHBA-AS1, MIR4435-2HG, and CEBPA-AS1, are enriched in human gastric cancer tissues and significantly elevated in the plasma of patients with gastric cancer. These findings indicate that the combination of these four lncRNAs might be used as diagnostic or prognostic markers for gastric cancer patients.

Keywords: RNA fragments; biomarker; gastric cancer; long noncoding RNA; plasma.

Figure 1. Gene expression levels in gastric tissues

Expression level of lncRNA INHBA-AS1 (A), MIR4435-2HG (B), CEBPA-AS1 (C), UCA1 (D), and AK001058 (E) in 49 paired gastric tissues. -ΔΔCt= (Ct_{target gene in normal}-Ct_{18S rRNA in normal}) - (Ct_{target gene in cancer}-Ct_{18S rRNA in cancer}).

Figure 2. Distribution of lncRNAs in plasma and AUC for lncRNA panels in training set

Distribution of lncRNA-INHBA-AS1 (A), MIR4435-2HG (B), CEBPA-AS1 (C), UCA1 (D), and AK001058 (E) levels in the plasma from patients and healthy controls in the training set. ΔCt_{target gene in cancer}= Ct_{target gene in cancer}-Ct_{18S rRNA in cancer}; ΔCt_{target gene in normal}= Ct_{target gene in normal}-Ct_{18S rRNA in normal}. And the ROCs of the 5 lncRNA (F), 5-lncRNA and five-minus-one lncRNA (G), and a new 4-lncRNA signature and four-minus-one lncRNA (H) in training set expressed in plasma, respectively. The AUCs and p-values are listed in the picture.

Figure 3. The relationship between lncRNA levels and the clinicopathological features of GC patients in training set

The relationship between the expression level of INHBA-AS1 and tumor size (A) and tumor grade (B); the relationship between the expression level of AK001058 and the depth of tumor invasion (C) and TNM stage (D). p-values are listed in the picture. ΔCt_{target gene in cancer}= Ct_{target gene in cancer}-Ct_{18S rRNA in cancer}.

Figure 4. Distribution of lncRNAs in plasma and AUC for lncRNA panels in testing set

Distribution of lncRNA-INHBA-AS1 (A), MIR4435-2HG (B), CEBPA-AS1 (C) and AK001058 (D) levels in the plasma from patients and healthy controls in the testing set. ΔCt_{target gene in cancer}= Ct_{target gene in cancer}-Ct_{18S rRNA in cancer}; ΔCt_{target gene in normal}= Ct_{target gene in normal}-Ct_{18S rRNA in normal}. The ROCs of the 4 lncRNA (E), 4-lncRNA and four-minus-one lncRNA (F) expressed in plasma in testing set. The AUCs and p-values are listed in the picture.

Figure 5. Expression of diverse MIR4435-2HG fragments in health control and gastric cancer plasma

(A) 13 different primer pairs of MIR4435-2HG gene, a-m fragments are the qRT-PCR products with different length. a: 186–291(106 bp); b: 424–529(106 bp); c: 728–833(106 bp); d: 1035–1151(117 bp); e: 1257–1356(100 bp);f: 1530–166(136bp); g: 1955–2079(125 bp); h: 2270-2359(90 bp); i: 2609–2692(84 bp); j: 2716–2841(126 bp); k: 3164–3273(110 bp); l: 3319-3457(139bp); m: 3657–3791(135 bp). (B) Expression level of MIR4435-2HG fragments in three GC patient and health control samples relative to 18S rRNA. Fragment i, j cannot be detected among the six plasma samples, and fragment l can be detected excluding one sample of three healthy controls.