Identification And Preliminary Validation Of Abnormal Chromosomal Changes And Related Genes In Rhabdomyosarcoma

Part I Identification and analysis of abnormal chromosomal changes and related genes in rhabdomyosarcomaObjective Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma with poor prognosis. However, the genetic etiology of RMS underlying its development and progression remains largely unclear. This study aims to reveal chromosome fragment and identify genes associated with rhabdomyosarcoma based on our previous studies. Another aim of this study is to perform bioinformatics-based functional enrichment analysis for genes in the genomic regions with copy number variations (CNVs). The author believes that this study would lay a foundation for the mechanism, molecular diagnosis and targeted therapy of RMS.Methods We used high-resolution array comparative genomic hybridization (aCGH) to explore tumor-associated CNVs and genes in20cases of RMS tissues and2cases of RMS cell lines. We then performed bioinformatics-based functional enrichment analysis using the DAVID software 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 using the TAM software.Results (1) aCGH analyses revealed that all RMS tissues and cell lines showed specific gains and losses. The recurrent regions with gains were12ql3.3-q14.1(12/20,60%),12q14.1(12/20,60%), and12q13.3(11/20,55%). The recurrent regions with losses were14q32.33(12/20,60%),9p12-p11.2(11/20,55%),10q11.21-q11.22(11/20,55%), and16p11.2(11/20,55%). The amplification regions were12q13.3-q14.1(6/20,30%), and12q13.3(5/20,25%). The deletion regions were9p24.3(8/20,40%),5q13.2(7/20,35%), and22q11.21(7/20,35%). The recurrent regions with gains were7q11.23(6/10,60%),19q13.12(6/10,60%),19p13.11(6/10,60%), and8q24.3(6/10,60%) in ERMS (embryonal RMS), while they rarely appeared or even did not appear in ARMS (alveolar RMS).(2) Bioinformatic analysis showed that the representative amplification genes were related to the immunoglobulin domain, IgG binding, receptor, and Rho-GAP domain in RMS. The representative deletion genes were related to defensin, wound healing, GTPase activation, and Fork-head DNA-binding region in RMS. The representative amplification genes were related to cell cycle process, proto-oncogene, cell cycle phase in ARMS by functional annotation clustering, while functional annotation clustering of the cell cycle process included MDM2, CDK4, and HMGA2genes and functional annotation clustering of proto-oncogene included GLI1, MDM2, CDK4, and c-MET genes. The upregulation of onco-miRNA, cell cycle-related miRNA, and muscle development miRNA were associated with RMS. The regulation of muscle development miRNAs included miR-24, miR-27a, and miR-331. The onco-miRNAs included miR-24, miR-27a, and miR-146b.Conclusions (1)12ql3.3-ql4.1,12ql4.1,12ql3.3,14q32.33,10q11.21-q11.22, and9p24.3are the most common chromosomal abnormalities fragment in RMS, while the regions of7q11.23,19q13.12,19p13.11, and8q24.3have association with ARMS.(2) Cell cycle process and proto-oncogene functional annotation clustering containing MDM2, GLI1, GEFT, CDK4and c-MET gene may be related with the occurrence and development of RMS.(3) The upregulation of onco-miRNA, cell cycle-related miRNA, and muscle development miRNA may be associated with RMS. Part Ⅱ The expression and roles of GEFT, CDK4, GLI1, MDM2, and c-MET genes in RMSObjective Based on the aCGH study, to detect and preliminarily validate the expression of GEFT, CDK4, GLI1, MDM2, and c-MET gene in RMS and their clinic pathological significance for RMS, which aims to lay a foundation for further elucidating the molecular mechanism, molecular diagnosis and targeted therapy of RMS.Methods Quantitative real-time polymerase chain reaction (QRT-PCR) was utilized to investigate the mRNA expression of GEFT, CDK4, GLI1, MDM2, and c-MET in RMS and normal control tissues. We applied immunohistochemistry to determine the protein expression of GEFT and CDK4for the45cases of RMS and36cases of normal control tissues, which were then combined with the clinical pathological and follow-up data for exploring their significance in RMS.Results (1) The expression levels of GEFT, CDK4, GLI1, MDM2and c-MET mRNA in RMSs were significantly higher than those in the normal control group by QRT-PCR. The mean mRNA level of GEFT, CDK4, GLI1, MDM2and c-MET in RMS samples was3.92-fold,4.53-fold,5.672-fold,14.59-fold,4.925-fold higher than that in the controls (p=0.0354, p=0.0061, p=0.0478, p=0.0229, p=0.0492, respectively), respectively.(2) Using immunohistochemistry, we found that the overexpression rate of GEFT in RMS samples was68.89%(31/45). The overexpression of GEFT was significantly correlated with advanced disease stages (p=0.001), lymph node metastasis (p=0.019), distant metastasis (p=0.004), and poor prognosis (p=0.001) of RMS. The expression rate of CDK4in RMS samples was48.89%(22/45). The expression of CDK4was significantly different between ARMS and ERMS (p=0.048). CDK4protein was more likely to be expressed in ARMS. The expression of CDK4was significantly correlated with lymph node metastasis in RMS (p=0.026).Conclusions (1) GEFT, CDK4, GLI1, MDM2and c-MET may be associated with RMS.(2) GEFT and CDK4may be closely associated with the progression, invasion, metastasis and poor prognosis of RMS.