Identification of novel long non-coding RNAs in clear cell renal cell carcinoma

Abstract

Background: Long non-coding RNAs (lncRNA) play an important role in carcinogenesis; knowledge on lncRNA expression in renal cell carcinoma is rudimental. As a basis for biomarker development, we aimed to explore the lncRNA expression profile in clear cell renal cell carcinoma (ccRCC) tissue.

Results: Microarray experiments were performed to determine the expression of 32,183 lncRNA transcripts belonging to 17,512 lncRNAs in 15 corresponding normal and malignant renal tissues. Validation was performed using quantitative real-time PCR in 55 ccRCC and 52 normal renal specimens. Computational analysis was performed to determine lncRNA-microRNA (MiRTarget2) and lncRNA-protein (catRAPID omics) interactions. We identified 1,308 dysregulated transcripts (expression change >2-fold; upregulated: 568, downregulated: 740) in ccRCC tissue. Among these, aberrant expression was validated using PCR: lnc-BMP2-2 (mean expression change: 37-fold), lnc-CPN2-1 (13-fold), lnc-FZD1-2 (9-fold), lnc-ITPR2-3 (15-fold), lnc-SLC30A4-1 (15-fold), and lnc-SPAM1-6 (10-fold) were highly overexpressed in ccRCC, whereas lnc-ACACA-1 (135-fold), lnc-FOXG1-2 (19-fold), lnc-LCP2-2 (2-fold), lnc-RP3-368B9 (19-fold), and lnc-TTC34-3 (314-fold) were downregulated. There was no correlation between lncRNA expression with clinical-pathological parameters. Computational analyses revealed that these lncRNAs are involved in RNA-protein networks related to splicing, binding, transport, localization, and processing of RNA. Small interfering RNA (siRNA)-mediated knockdown of lnc-BMP2-2 and lnc-CPN2-1 did not influence cell proliferation.

Conclusions: We identified many novel lncRNA transcripts dysregulated in ccRCC which may be useful for novel diagnostic biomarkers.

Figure 1

lncRNA expression profiling. The expression of 32,183 lncRNA transcripts was determined in 15 corresponding clear cell renal carcinoma (ccRCC) and normal renal tissue samples. The expression profile of 25 different lncRNA transcript variants allows accurate discrimination of normal and malignant renal tissue as shown in the heatmap. All ccRCC tissues (green bar above the heatmap) and normal renal tissues (red bar) are located in a separate cluster.

Figure 2

Validation of lncRNA dysregulation. The expression of 13 target lncRNAs in renal cell carcinoma (red) and normal renal (green) tissue was validated using quantitative real-time PCR; the expression levels were normalized using ACTB and PPIA as reference genes. We confirmed significant (all p < 0.001) overexpression of lnc-BMP2-2 (B), lnc-CPN2-1 (C), lnc-FZD1-2 (F), lnc-ITPR2-3 (G), lnc-SLC30A6-1 (K), and lnc-SPAM1-6 (L) in renal cell carcinoma, whereas lnc-ACACA-1 (A), lnc-FOXG1-2 (E), lnc-LCP2-2 (H), lnc-RP3-368B9 (I), and lnc-TTC34-3 (M) were significantly downregulated (all p < 0.001); lnc-ERCC5-1 (D, p = 0.401) and lnc-RP11-480I12.4.1-1 (J, p = 0.731) were not different in malignant and normal renal tissue. Receiver operator characteristic (N) analyses demonstrate excellent discrimination of the lncRNAs between both cohorts (area under the curve 0.90–0.94).

Figure 3

lncRNA expression after siRNA-mediated lncRNA knockdown. Expression of lnc-BMP2-2 (A) and lnc-CPN2-1 (B) was manipulated using specific siRNA in Caki-1, Caki-2, and A-498 renal cell carcinoma cell lines. A-498 did not express lnc-BMP2-2. lnc-BMP2-2 expression was significantly (p < 0.05) decreased in siRNA-treated Caki-1 and Caki-2 cells; lnc-CPN2-1 was also decreased in Caki-2 cells.

Figure 4

Cell proliferation after siRNA-mediated lncRNA knockdown. The EZ4U test was applied for cell proliferation testing; proliferative activity was not changed in any renal cell carcinoma cell line (A, A-498; B, Caki-1; C, Caki-2).