Expression pattern of long non-coding RNAs in renal cell carcinoma revealed by microarray

Abstract

Background: Recent large-scale transcriptome analyses have found large numbers of transcripts, including that of long non-coding RNAs (lncRNAs), which are aberrant in various diseases, especially cancers. However, it is not clear whether lncRNAs are involved specifically in renal cell carcinoma (RCC). We investigated the expression patterns of lncRNAs in five RCC tumor samples (T) relative to those of matched adjacent non-tumor tissues (N) via microarray.

Methods: A microarray with 33,045 lncRNA probes and 30,215 mRNA probes was used to identify deregulated lncRNAs in five RCC patients. Furthermore, we confirmed the relative expression levels of AK096725 and ENST00000453068 in 70 paired samples by quantitative reverse transcription polymerase chain reaction (qRT-PCR).

Results: The lncRNA microarray revealed 27,279 lncRNAs in RCC samples, of which 480 were significantly upregulated (P<0.05; T/N>1.5) and 417 were significantly downregulated (P<0.05; N/T>1.5) compared with the matched non-tumor samples. In addition, 19,995 mRNAs were detected, of which 458 were significantly upregulated (P<0.05; T/N>1.5) and 413 were significantly downregulated (P<0.05; N/T>1.5). The expression level changes of AK096725 (P = 0.043) and ENST00000453068 (P<0.001) in 70 paired samples were in accord with the microarray data.

Conclusions: The study uncovered expression patterns of lncRNAs in 5 RCC patients, as well as a number of aberrant lncRNAs and mRNAs in tumor samples compared with the non-tumor tissues. The revelation of an association between AK096725 expression and RCC is especially noteworthy. These findings may help to find new biomarkers in RCC.

Figure 1. Overview of the microarray signatures.

(A and C) Scatter-plots showing the variations in (A) the lncRNA and (C) protein-coding mRNA expressions between the tumor and non-tumor matched pairs of tissues. The values of the X and Y axes are the averaged normalized signal values of groups of samples (log₂ scaled). The green lines are fold change lines (FC = 1.5). The color of the points indicates the intensities from low (blue) to high (red). The lncRNAs or mRNAs above the top green line and below the bottom green line indicated >1.5 FC between the two groups of samples. (B and D) Volcano plots of the differentially expressed (B) lncRNAs and (D) protein-coding mRNAs. The vertical lines correspond to 1.5 FC up and down and the horizontal line represents a P-value of 0.05. The red point in the plot represents the differentially expressed lncRNAs or mRNAs with statistical significance.

Figure 2. Relative expressions of AK096725 and ENST00000453068 in 70 paired RCC tumor specimens and adjacent normal tissue samples determined by qRT-PCR.

The expression level of lncRNAs was normalized using β-actin as an internal control. The median in each triplicate was used to calculate the relative lncRNA concentration using the comparative ΔCt method. The lines are at mean with SEM. AK096725 was found significantly differentially upregulated in tumor specimens (P = 0.043), while ENST00000453068 was identified as significantly downregulated in tumor specimens (P<0.001).

Figure 3. Analysis of AK096725 mRNA expression levels from the tumor and non-tumor tissues of ccRCC patients (57 cases) and nccRCC patients (13 cases), from qRT-PCR.

β-actin was used as a control. Data is shown as fold change which is the normalized expression of tumor and non-tumor tissue from the same patient. The lines are at mean with SEM. The mRNA expression of AK096725 of ccRCC patients was significantly lower than that of the nccRCC patients (P<0.001).