Identification of the Different Roles and Potential Mechanisms of T Isoforms in the Tumor Recurrence and Cell Cycle of Chordomas

Abstract

Purpose: The roles of T (brachyury) isoforms in chordomas remain unclear. This study aimed to investigate the different roles and mechanisms of them in chordomas.

Patients and methods: The expression of T isoforms mRNAs in 57 chordomas was assessed, and a prognosis analysis was conducted. Cell apoptosis, proliferation and cell cycle assays were performed after specific T isoform mRNA knockdown. Whole-transcriptome sequencing, Gene Set Enrichment Analysis, Gene Ontology analysis, Kyoto Encyclopedia of Genes and Genomes analysis and competing endogenous RNA (ceRNA) analysis were conducted.

Results: As revealed in this study, the T-long isoform was a significant risk factor (hazard ratio [HR], 1.09; P=0.018) and the T-short isoform was a protective factor (HR, 0.24; P=0.012) associated with tumor recurrence. After T-long isoform knockdown, the cell cycle was arrested at G0/G1 phase and cell proliferation was significantly inhibited. A bioinformatic analysis revealed that the upregulation of H19, P21 and GADD45B; downregulation of SKP2 and CDK2; and accompanying changes in the P53 signaling pathway consistently contributed to G0/G1 arrest. After T-short isoform knockdown, the cell cycle was arrested at G2/M phase and cell apoptosis tended to increase slightly (P=0.067). The upregulation of YWHAZ and downregulation of E2F1 and its target genes might contribute to cell cycle arrest in G2/M phase and apoptosis. In addition, the ceRNA network, consisting of long noncoding RNAs, mRNAs and microRNAs, was established.

Conclusion: The T-long isoform was a risk factor and the T-short isoform was a protective factor for chordoma recurrence. In addition, the cell cycle was the main target of T isoforms knockdown, and the changes in the downstream transcriptome may contribute to the different effects of specific T isoform knockdown on the changes in the cell cycle distributions and apoptosis and proliferation of chordoma cells.

Keywords: brachyury; ceRNA; cell cycle; chordoma; prognosis; whole-transcriptome sequencing.

Figure 1

Targets, knockdown efficacy and specificity of siRNAs targeting specific T isoforms. (A) The T-long isoform siRNA was designed to mainly bind to exon 7 of the T-long isoform mRNA. The T-short isoform siRNA was designed to bind the junction of exon 6 and exon 8 in the T-short isoform mRNA, which was a continuous sequence in the T-short isoform mRNA. (B) The qPCR assay confirmed the knockdown efficacy and specificity of siRNAs targeting specific T isoforms. LongTKD: T-long isoform knockdown; ShortTKD: T-short isoform knockdown.

Figure 2

Cell apoptosis, proliferation and cell cycle changes after the knockdown of specific T isoforms. (A–D) Early apoptotic cells labelled with PE-Annexin V but not 7-AAD. The percentage of early apoptotic cells (Q3) was slightly increased after T-short isoform knockdown (ShortTKD) compared with the negative control (NC) (P=0.067). (E–H) Proliferating cells were labelled with BrdU. Proliferation (BrdU labeling index) was significantly inhibited after T-long isoform knockdown (LongTKD). (I–N) Analysis of the cell cycle after labeling with BrdU and 7-AAD. A significantly greater percentage of cells in G0/G1 phase was observed after LongTKD than in the NC. A significantly lower percentage of cells in S phase was observed after LongTKD. A significantly greater percentage of cells in G2/M phase was observed after ShortTKD.

Figure 3

Gene set enrichment analysis (GSEA) analysis, differentially expressed (DE) genes and enrichment analysis. (A, B) GSEA of hallmark gene sets after T-long isoform knockdown (LongTKD). Interferon alpha response, xenobiotic metabolism, myogenesis and P53 pathway gene sets were significantly enriched upregulated gene sets. Additionally, E2F targets, G2/M checkpoint, protein secretion and mitotic spindle gene sets were the top four enriched downregulated gene sets. (C) GSEA of hallmark gene sets after T-short isoform knockdown (ShortTKD). The myogenesis gene set was the top enriched upregulated gene set. E2F targets, G2/M checkpoint and TNFa signaling via NF-kB gene sets were the top three significantly enriched downregulated gene sets. (D) Venn plots of DE genes. In the comparison between LongTKD and NC, 1479 DE protein-coding RNAs (pcRNAs, 892 downregulated), 8 DE microRNAs (miRNAs, 7 downregulated) and 115 DE long noncoding RNAs (lncRNAs, 56 downregulated) were identified. In the comparison between ShortTKD and NC, 636 DE pcRNAs (342 downregulated), 4 DE miRNAs (3 downregulated) and 15 DE lncRNAs (12 downregulated) were identified. (E). GO enrichment analysis of DE genes after LongTKD. G1/S phase transition, DNA replication, chromosome and sister chromatid segregation were the main enriched terms in the biological process (BP) category, while condensed chromosome, kinetochore, and membrane coat were the main enriched terms in the cellular component (CC) category. (F) GO enrichment analysis of DE genes after ShortTKD. Cell cycle G2/M phase transition, Ras protein signal transduction, regulation of locomotion, protein K48-linked ubiquitination and other terms significantly enriched in the BP category, while midbody, cell-cell junction, membrane contact site were significantly enriched CC terms. However, no significantly enriched terms were identified in the MF category. (G) KEGG enrichment analysis of DE genes after LongTKD. Cell cycle, the P53 signaling pathway and PI3K-Akt signaling pathway (q = 0.083) were enriched. (H) KEGG enrichment analysis of DE genes after ShortTKD. The ErbB signaling pathway and tight junction were enriched.

Figure 4

ceRNA regulatory network after T-long isoform knockdown. The lncRNA (long noncoding RNA) H19 interacted with 12 miRNAs (microRNAs), while KCNQ1OT1 interacted with 18 miRNAs. Additionally, 19 pcRNAs (protein-coding RNAs) were downregulated and 45 pcRNAs were upregulated. The pcRNA NFIB interacted with 11 miRNAs, while CALU interacted with 10 miRNAs. Moreover, miR-29a-3p, miR-29b-3p and miR-29c-3p interacted with more than 30 pcRNAs or lncRNAs. Notably, miR-130a-3p, miR-130b-3p and miR-454-3p participated in more than 20 interactions with pcRNAs or lncRNAs. Additionally, miR-3666, miR-4295, miR-148b-3p, miR-152-3p, miR-148a-3p participated in 10 interactions with pcRNAs or lncRNAs.