Genome-wide analysis of long noncoding RNA (lncRNA) expression in colorectal cancer tissues from patients with liver metastasis

Abstract

The liver is the most frequent site of metastasis in colorectal cancer (CRC), in which long noncoding RNAs (lncRNAs) may play a crucial role. In this study, we performed a genome-wide analysis of lncRNA expression to identify novel targets for the further study of liver metastasis in CRC. Samples obtained from CRC patients were analyzed using Arraystar human 8 × 60K lncRNA/mRNA v3.0 microarrays chips to find differentially expressed lncRNAs and mRNAs. The results were confirmed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The differentially expressed lncRNAs and mRNAs were identified through fold change filtering. Gene ontology (GO) and pathway analyses were performed using standard enrichment computational methods. In the CRC tissues from patients with liver metastasis, 2636 lncRNAs were differentially expressed, including 1600 up-regulated and 1036 down-regulated over two-fold compared with the CRC tissues without metastasis. Among the 1584 differentially expressed mRNAs, 548 were up-regulated and 1036 down-regulated. GO and pathway analysis of the up-regulated and down-regulated mRNAs yielded different results. The up-regulated mRNAs were associated with single-organism process (biological process), membrane part (cellular component), and transporter activity (molecular function), whereas the down-regulated mRNAs were associated with cellular process, membrane, and binding, respectively. In the pathway analysis, 27 gene pathways associated with the up-regulated mRNAs and 51 gene pathways associated with the down-regulated mRNAs were targeted. The significant changes in NQO2 (NM_000904) mRNA and six associated lncRNAs were selected for validation by qRT-PCR. Aberrantly expressed lncRNAs may play an important role in the liver metastasis of CRC. The further study can provide useful insights into the biology and, ultimately, the prevention of liver metastasis.

Keywords: NAD (P) H Dehydrogenase, Quinone 2 (NQO2); colorectal cancer (CRC); long noncoding RNA (lncRNA).

Figure 1

Differentially expressed long noncoding RNAs (lncRNAs) and mRNAs in colorectal cancer (CRC) tissues from patients with Liver Metastasis (Exp G) and without metastasis (Ctrl G). Hierarchical cluster analysis of CRC samples to assess the significant expression of lncRNAs (A1) and mRNAs (A2) based on microarray analyses. Red denotes high relative expression and green low relative expression. Each RNA is represented by a single row of colored boxes and each sample by a single column. The box plot shows the variations in lncRNA (B1) and mRNA (B2) expression. The scatter plot and the volcano plot illustrate the distributions of the data in the lncRNA (C1, D1) and mRNA (C2, D2) profiles. After data normalization, the distributions of the log₂ ratios among samples were nearly the same. The values of the x‐ and y‐axes in the scatter plot were the averaged normalized signal values of the group (log₂ scaled). The green lines in the scatter and volcano plots show the significant fold change.

Figure 2

Gene ontology (GO) enrichment analysis of lncRNA‐target genes. GO analysis of lncRNA‐target genes according to biological process (A), cell component (B) and molecular function (C), including analyses of the up‐regulated (A1, B1 and C1) and down‐regulated (A2, B2 and C2) lncRNAs.

Figure 3

The top 10 enrichment scores in the pathway analysis of the up‐regulated (A) and down‐regulated (B) mRNAs. The lower panel shows the leading pathways associated with the up‐regulated (C) and down‐regulated (D) mRNAs.

Figure 4

Predictions of the coding‐noncoding gene co‐expression network. The co‐expression network was composed of one mRNA and 769 potentially associated lncRNAs. The 340 direct connections, indicated by the red solid line, are those in which the correlation between the mRNA and the potentially associated lncRNAs was positive. The 429 direct connections, indicated by the green solid line show the respective of negative correlations.

Figure 5

Quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR) validation of 10 randomly selected differentially expressed lncRNAs. Six of the qRT‐PCR‐validated lncRNAs (NR_046711, NR_036537, NR_036580, NR_002795, NR_033878, and NR_036484) showed the same fold change tendencies as those in the microarray results (*P < 0.05). The differences in the other four qRT‐PCR‐validated lncRNAs (NR_034129, NR_004855, NR_027054 and NR_027242) were not statistically significant. (A) Comparison of the microarray data and qPCR results; (B) Comparison of colorectal cancer tissues from patients with Liver Metastasis (Exp G) and without metastasis (Ctrl G).