Screening And Validation Of Possible Relative Oncogenes/Tumor Suppressor Genes In Nasopharyngeal Carcinoma Cell Lines

Nasopharyngeal carcinoma(NPC)is a common epithelial tumor, the vast majority of which is belonged to poorly differentiated squamous cell carcinoma with high malignant degree. NPC has an extremely uneven geographical distribution and is a high incidence of malignancy in Southern China and Southeast Asia, especially those of Cantonese origin.its etiologies are associated with genetic susceptibility, Epstein-Barr virus(EBV)infection and environmental factors. Both genetic(such as gene amplification, deletion and mutation)and epigenetic(methylation) changes contribute to this process by altering the functions of the genes in their critical roles in cell proliferation, apoptosis, genome stability and differentiation of the nasopharyngeal epithelial cells. However, the molecular mechanisms of NPC pathogenesis are still unknown.NPC carcinogenesis is believed to be a multistep process whose progression involved in the abnormal activation of oncogenes and loss of tumor suppressor genes. Oncogenes come from the activation of proto-oncogenes that exist normal epithelial cells, including increase of number, activity and mutation. Tumor suppressor genes can block cell proliferation, promote cell differentiation in normal cells. The loss and mutation of tumor suppressor genes will lead oncogenes to produce a marked effect which can result in the development of NPC.Oncogenes and tumor suppressor genes also play a critical role in the development of NPC. So far, significant progress in the study of tumor suppressor gene in NPC has been made, such as RASSF1A±BLU,BRD7,DAPK1,DLC1 gene et al, more than 30 genes has been proven as candidate tumor suppressor genes of NPC because of gene silencing on biological behaviors of NPC cells, and promoter methylation is the primary molecular mechanism. However, the study on oncogenes in NPC relatively lags behind. Due to amplification or over-expression in NPC, BCL2,CCND1,EGFR,TP73L,EVI1,HER2,HRAS,NRAS,STAT1,STAT2,INT2/FGF3,MDM2,MYC,PIK3CA genes et al have been speculated as candidate oncogenes of NPC, but rarely has been reported impact of gene over-expression on biological activity of NPC, nor studies in vivo reported. Therefore, to study on tumor suppressor genes in NPC, particularly oncogenes, And moreover, to study on function and mechanism of genes implicated in NPC carcinogenesis and development,which have made significant importance in the development of NPCArray technique provides very good platform for the screening of candidate genes. Because of the characteristic including stabilization, high-flux, high efficiency and little samples can satisfy the need of differential expression genes research between several specimens, so it is generally used to molecular biology research of tumor. Previously, we have constructed tree model for NPC carcinogenesis using 170 comparative genomic hybridization(CGH)samples data from five studies. The tree model reveals 11 important events in NPC and suggested that the amplification region within 12p12-11 may also be an early event during NPC carcinogenesis. But there are hundreds of genes within this region.2005,a paper in Nature found the oncogene MITF in melanoma through intergrating genomic and transcriptomic mapping data. Early day, we screened amplification and over-expression gene, PTHLH, within 12p12-11 in NPC with the same way. PTHLH is a family of parathyroid hormone receptor, it plays a important role in the origin and progression of many tumors such as Humoral hypercalcemia of malignancy, breast carcinoma, prostatic carcinoma, colon carcinoma etc. So we presume that PTHLH may be participated in origin and progression of NPC.Based on the work we have done, the purpose of our topic is to screen the candidate oncogenes and tumor suppressor genes in NPC cell lines(CNE1,CME2,HONE1)with the intergrating analytical method, predict the possibly function of candidate oncogenes and tumor suppressor genes by bioinformatics. Then,we change the expression level of PTHHLH in NPC cell line, observe biological behaviors of NPC cell line and the potential mechanism of action. The identification of its characteristics in NPC cells will help us understand it's contributes to tumorigenesis of NPC.Therefore, we did these researches as follow:1 Screening and bioinformatics prediction of candidate oncogenes and tumor suppressor genes in NPCCandidate oncogenes and tumor suppressor genes in NPC were screened through integrating SNP array, gene expression profiles microarray and copy number variable (DNVs)data by Ensembl database. We got 160 target genes, including 102 amplification and over-expression genes and 58 deletion and low-expression genes. For verification the reliability of array, we randomly choose 9 target genes and test their expression level with quantitive real-time PCR. The result showed coincidence between QRT-PCR data and microarray data except ILK. Bioinformatics on web was used to predict the function of target genes. They have various kinds of functions including transcription factor transport protein, regulatory protein, biologic enzyme, kinase etc and participate many pathways such as P53 pathway, cell cycle and apoptosis pathway etc. All of these biological functions were associated with traits that were the hallmarks of the malignant phenotype, with a focus on tumor-host interactions. Accordingly, the disorder of candidate oncogenes and tumor suppressor genes expression might lead to the changes of pathways and multiple cell regulatory mechanisms in NPC.In addition, we did cluster analysis and network diagram of the three group datas. All of them were associated with oncogenes and tumor suppressor genes, Epithelial-mesenchymal transition(EMT),NPC and EBV. In order to improve these datas were not stochastic production, we did 10,000 stochastic simulation. The results have significantly deviation(P=0.008,0.044,0.006)Sebsequently, we overexpressed candidate gene PTHLH that we had proved before and examined the role of PTHLH in NPC cell line. Meanwhile, based on bioinformatics analyses, we predicted genes that may be involved in PTHLH signaling pathway.2 Effects of PTHLH overexpression on biological behaviors of CNE1 cellsQuantitive real-time PCR was used to detect the expression of PTHLH in NPC-derived cell lines(5-8F,6-10B, CNE1,CNE2 and HONE1) and immortalized nasopharyngeal epithelial cells NP69. The results of one way ANOVA analysis showed that the expression of PTHLH in 6 cell lines was significantly different from each other (F=100.473,P=0.000).The results of 2-ΔΔCt method showed the expression of PTHLH was significantly upregulated in 5-8F,6-10B, CNE1,CNE2 and HONE1 as compared with NP69, but with the lowest expression in CNE1. Thus,CNE1 was choosed to as the over-expression cell line.Approximately 528bp PTHLH gene segments were amplified from genomic locus and inserted to a PGC-FU vector containing GFP. And then NPC cell line CNE1 was transfected with the vector pGC-FU/PTHLH and then separated the cells with GFP. Lastly, a NPC cell line CNE1-pGC-FU/PTHLH was established, with stable overexpression of PTHLH. CNE1-pGC-FU/PTHLH cells showed a significantly increased proliferation compared with CNE1 and CNE1-pGC-FU cells as determined by in vitro MTT assay (F=1208.480,P=0.000).In addition, CNE1-pGC-FU/PTHLH cells had a significant increase in their ability to form colonies in plate as compared with CNE1 and CNE1-pGC-FU cells(F=43.771,P=0.000).Besides, the cell cycle G1 and S distribution detected by flow cytometry is also significantly different from each other (F=12.722,P=0.002)(F=42.888,P=0.000).These results indicated overexpression of PTHLH significantly increased proliferation of CNE1 cells.The results of Corning Transwell Inserts showed that CNE1-pGC-FU/PTHLH cells had significantly increased motility as compared with CNE1 and CNE1-pGC-FU cells(F=316.382,P=0.000).Furthermore,CNE1-pGC-FU/PTHLH cells also had significantly inhenced motility as compared with CNE1 and CNE1-pGC-FU cells by use of scracthing. All of these results showed overexpression of PHTLH partially led to upregulation motility of CNE1 cells.CDK2, one of candidate oncogenes may be react in the same pathway with PTHLH, was detected by RT-PCR, the result showed that the expression in CNE1-pGC-FU/PTHLH cells had significantly increased as compared with CNE1 and CNE1-pGC-FU cells(F=5.527,P=0.024)Conclusions1.Screening and bioinformatics prediction of relative oncogenes and tumor suppressor genes in NPC2.These target genes have close relation with EBV and EMT and maybe join the development of NPC3.Overexpression of PTHLH in vitro results in a significant increase of cell proliferation, motility etc in NPC cell lines CNE1...