Long Non-Coding PROX1-AS1 Expression Correlates with Renal Cell Carcinoma Metastasis and Aggressiveness

Abstract

Long non-coding RNAs (lncRNAs) can be specifically expressed in different tissues and cancers. By controlling the gene expression at the transcriptional and translational levels, lncRNAs have been reported to be involved in tumor growth and metastasis. Recent data demonstrated that multiple lncRNAs have a crucial role in renal cell carcinoma (RCC) progression-the most common malignant urogenital tumor. In the present study, we found a trend towards increased PROX1 antisense RNA 1 (PROX1-AS1) expression in RCC specimens compared to non-tumoral margins. Next, we found a positive correlation between PROX1-AS1 expression and the occurrence of distant and lymph node metastasis, higher tumor stage (pT1 vs. pT2 vs. pT3-T4) and high-grade (G1/G2 vs. G3/G4) clear RCC. Furthermore, global demethylation in RCC-derived cell lines (769-P and A498) and human embryonic kidney 293 (HEK293) cells induced a significant increase of PROX1-AS1 expression level, with the most remarkable change in HEK293 cells. In line with this evidence, bisulfite sequencing analysis confirmed the specific demethylation of bioinformatically selected CpG islands on the PROX1-AS1 promoter sequence in the HEK293 cell line but not in the tumor cells. Additionally, the human specimen analysis showed the hemimethylated state of CG dinucleotides in non-tumor kidney tissues, whereas the tumor samples presented the complete, partial, or no demethylation of CpG-islands. In conclusion, our study indicated that PROX1-AS1 could be associated with RCC progression, and further investigations may define its role as a new diagnostic marker and therapeutic target.

Keywords: PROX1-AS1; human specimens; non-coding RNA; renal cell carcinoma.

Figure 1

The PROX1 antisense RNA 1 structure (PROX1-AS1). The PROX1-AS1 presents a transcript of 3399 bp, including six exons with a faint (6.18%) probability of protein translation (according to the Coding-Potential Assessment Tool (CPAT)). LncRNA-PROX1-AS1 is located on the long arm of chromosome 1 at position 1q32.3 (start position: 213820404; end position: 213987547) in the regions AC096639.2-AC011700.5 of the DNA segments. This information is available at LNCipedia [29], RNA central [30], and Ensemble [31] databases.

Figure 2

Expression of PROX1-AS1 in human renal cell carcinomas. (A) PROX1-AS1 expression is higher in patients with metastases (M1) compared to patients without metastasis (M0). (B) Increased PROX1-AS1 expression corresponded to lymph node tumor infiltration. (C) PROX1-AS1 increased in the higher tumor stages (pT1 vs. pT2 and pT3–4). (D) PROX1-AS1 significantly increased in G3–G4 vs. G1–G2 grades in clear renal cell carcinomas. * p < 0.05.

Figure 3

PROX1-AS1 promoter region CpG island analysis. In the methylation simulation performed in the lncRNA-PROX1-AS1 exon 1 and the gene’s promoter (start position chr1: 213,985,153–end position: 214,000,000, accession data: >NC_000001.11:213985153-214000000 Homo sapiens chromosome 1, GRCh38.p13 Primary Assembly), three CpG islands (1: CpG 42; 2: CpG 84 and 3: CpG 40) were selected with UCSC Genome Browser (http://genome-euro.ucsc.edu/; accessed on 9 April 2021).

Figure 4

The demethylation induced by 5-aza-2′-deoxycytidine increased the expression of PROX1-AS1 in human renal carcinoma cells (769-P and A498) and embryonic kidney 293 cells (HEK293). PROX1-AS1 expression was determined by qRT-PCR following exposure of cells with 5 uM 5-aza-2′-deoxycytidine for 48 h. ** p ≤ 0.01, *** p ≤ 0.001.

Figure 5

CpG-islands 1 and 3 methylation in HEK293 before and after 5-aza-2′-deoxycytidine treatment. CpG island 1 and CpG island 3 before (top line) and after (bottom line) HEK293 treatment with 5-aza-2′-deoxycytidine. Arrows indicate the location of the demethylated cytosines after the treatment with the demethylating agent (the appearance of red peaks in the bottom line corresponds to cytosine demethylation).

Figure 6

Bisulfite sequencing of paired non-tumor and tumor tissues. CpG island 1: Top-line—hemimethylation in NT (blue and red peaks). Bottom line—differential methylation (blue or red peaks) in some CG in T. CpG island 3—hemimethylation in NT (blue and red peaks) and demethylation (only red peaks) in T.