Upregulation of 15 Antisense Long Non-Coding RNAs in Osteosarcoma

Abstract

The human genome encodes thousands of natural antisense long noncoding RNAs (lncRNAs); they play the essential role in regulation of gene expression at multiple levels, including replication, transcription and translation. Dysregulation of antisense lncRNAs plays indispensable roles in numerous biological progress, such as tumour progression, metastasis and resistance to therapeutic agents. To date, there have been several studies analysing antisense lncRNAs expression profiles in cancer, but not enough to highlight the complexity of the disease. In this study, we investigated the expression patterns of antisense lncRNAs from osteosarcoma and healthy bone samples (24 tumour-16 bone samples) using RNA sequencing. We identified 15 antisense lncRNAs (RUSC1-AS1, TBX2-AS1, PTOV1-AS1, UBE2D3-AS1, ERCC8-AS1, ZMIZ1-AS1, RNF144A-AS1, RDH10-AS1, TRG-AS1, GSN-AS1, HMGA2-AS1, ZNF528-AS1, OTUD6B-AS1, COX10-AS1 and SLC16A1-AS1) that were upregulated in tumour samples compared to bone sample controls. Further, we performed real-time polymerase chain reaction (RT-qPCR) to validate the expressions of the antisense lncRNAs in 8 different osteosarcoma cell lines (SaOS-2, G-292, HOS, U2-OS, 143B, SJSA-1, MG-63, and MNNG/HOS) compared to hFOB (human osteoblast cell line). These differentially expressed IncRNAs can be considered biomarkers and potential therapeutic targets for osteosarcoma.

Keywords: alternative splicing; antisense RNA; non-coding RNA; osteosarcoma; sarcoma.

Figure 1

Nuclear and cytoplasmic sense–antisense RNA pairing. Diagram of nuclear RNA duplex formation (A) that further results in alternative splicing (B) or RNA editing (C). Classification of sense/antisense pairs (D). Sense genes represent in blue, whereas antisense transcripts are pink. The black lines between the genes indicates regions of overlap. There are different types of natural antisense transcripts overlapping: head-to-head overlap in cis (Da), embedded overlap in cis (Db), tail-to-tail overlap in cis (Dc), and overlap in trans (Dd).

Figure 1

Nuclear and cytoplasmic sense–antisense RNA pairing. Diagram of nuclear RNA duplex formation (A) that further results in alternative splicing (B) or RNA editing (C). Classification of sense/antisense pairs (D). Sense genes represent in blue, whereas antisense transcripts are pink. The black lines between the genes indicates regions of overlap. There are different types of natural antisense transcripts overlapping: head-to-head overlap in cis (Da), embedded overlap in cis (Db), tail-to-tail overlap in cis (Dc), and overlap in trans (Dd).

Figure 2

3D Principal component analysis (PCA) clustered transcript expression profiling of tumour and normal samples. 3D-PCA plot highlights the 3 principal components (PC1, PC2 and PC3). The groups have been marked by different colour; blue: tumour, orange: normal. The figure highlights that the tumour and normal samples were clustered separately.

Figure 3

Heatmap of antisense lncRNAs expression comparison between the tumour (orange) and normal (green) samples.

Figure 4

Circos plot visualizes the antisense lncRNAs expression in individual samples. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 34, 35, 36, 37, 38, 39, 40 are tumour samples, whereas 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32 are normal samples. Each colour represents a transcript.

Figure 5

(A–G). RT-qPCR analysis of antisense lncRNAs. The transcripts expressions were determined using 8 different osteosarcoma cell line; SaOS-2, G-292, SJSA-1, HOS, 143B, U2-OS, MNNG/HOS, MG-63 and relative lncRNA expression obtained by hFOB expression. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 5

(A–G). RT-qPCR analysis of antisense lncRNAs. The transcripts expressions were determined using 8 different osteosarcoma cell line; SaOS-2, G-292, SJSA-1, HOS, 143B, U2-OS, MNNG/HOS, MG-63 and relative lncRNA expression obtained by hFOB expression. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 5

(A–G). RT-qPCR analysis of antisense lncRNAs. The transcripts expressions were determined using 8 different osteosarcoma cell line; SaOS-2, G-292, SJSA-1, HOS, 143B, U2-OS, MNNG/HOS, MG-63 and relative lncRNA expression obtained by hFOB expression. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.