Analysis of molecular cytogenetic alteration in rhabdomyosarcoma by array comparative genomic hybridization

Abstract

Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma with poor prognosis. The genetic etiology of RMS remains largely unclear underlying its development and progression. To reveal novel genes more precisely and new therapeutic targets associated with RMS, we used high-resolution array comparative genomic hybridization (aCGH) to explore tumor-associated copy number variations (CNVs) and genes in RMS. We confirmed several important genes by quantitative real-time polymerase chain reaction (QRT-PCR). We then performed bioinformatics-based functional enrichment analysis for genes located in the genomic regions with CNVs. In addition, we identified miRNAs located in the corresponding amplification and deletion regions and performed miRNA functional enrichment analysis. aCGH analyses revealed that all RMS showed specific gains and losses. The amplification regions were 12q13.12, 12q13.3, and 12q13.3-q14.1. The deletion regions were 1p21.1, 2q14.1, 5q13.2, 9p12, and 9q12. The recurrent regions with gains were 12q13.3, 12q13.3-q14.1, 12q14.1, and 17q25.1. The recurrent regions with losses were 9p12-p11.2, 10q11.21-q11.22, 14q32.33, 16p11.2, and 22q11.1. The mean mRNA level of GLI1 in RMS was 6.61-fold higher than that in controls (p = 0.0477) by QRT-PCR. Meanwhile, the mean mRNA level of GEFT in RMS samples was 3.92-fold higher than that in controls (p = 0.0354). Bioinformatic analysis showed that genes were enriched in functions such as immunoglobulin domain, induction of apoptosis, and defensin. Proto-oncogene functions were involved in alveolar RMS. miRNAs that located in the amplified regions in RMS tend to be enriched in oncogenic activity (miR-24 and miR-27a). In conclusion, this study identified a number of CNVs in RMS and functional analyses showed enrichment for genes and miRNAs located in these CNVs regions. These findings may potentially help the identification of novel biomarkers and/or drug targets implicated in diagnosis of and targeted therapy for RMS.

Figure 1. Genomic map of the aberrant regions in 20 cases human RMS chromosomes.

The first (outer) circle represents the human chromosome. From the second to the inner, circles highlight the gain regions in orange, the loss regions in purple, the amplification regions in red, and the deletion regions in green.

Figure 2. 12q13.3–q14.1 region are amplified in RMS.

X-axis: 12 chromosome; Y-axis: the sample chip hybridization signal Log2 ratio value. Fig. 2A. GLI1 is amplified in RMS. Fig. 2B. CDK4 is amplified in RMS.

Figure 3. Genomic map of the aberrant regions in a human RMS cell lines chromosomes.

The first (outer) circle represents the human chromosome. From the second to the inner, circles highlight the gain regions in orange, the loss regions in purple, the amplification regions in red, and the deletion regions in green. Fig 3A. PLA-802 cell line; Fig 3B. RD cell line.

Figure 4. Expression level of GLI1 and GEFT mRNA in RMS samples in comparison with normal muscle tissue.

Fig. 4A. Expression level of GLI1 mRNA in RMS compared with normal muscle tissue (3.421+1.034 vs 0.5174+0.083, p = 0.0477). Fig. 4B. Expression level of GEFT mRNA in RMS compared with normal muscle tissue (5.326+1.178 vs 1.359+0.294, p = 0.0354). Columns, the expression level of mRNA in whole RMS samples or normal muscle tissues; bars, SD.

Figure 5. Enriched functions of genes within the amplification regions (A) and deletion regions (B) in RMS.

Figure 6. Enriched functions of genes within the amplification regions (A) and deletion regions (B) in ARMS.

Figure 7. Enriched functions of genes within the amplification regions (A) and deletion regions (B) in ERMS.

Figure 8. Enriched miRNA functions of miRNAs within the amplification regions in RMS.