Integrated Genomic And Transcriptomic Analysis Identifies Candidate Oncogenes In Nasopharyngeal Carcinoma

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.The results of epidemiology showed NPC had a family history,which suggested genetic susceptibility might play an important role in the pathogenesis of NPC.Different etiologic factors such as early infection with Epstein-Barr virus(EBV),genetic predisposition,and chemical carcinogens in some traditional diets(particularly salted fish) have been associated with the development of NPC,the accumulation of genetic and epigenetics changes in tumor cells and drive the progressive transformation of normal human cells into invasive carcinoma.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 asopharyngeal epithelial cells.NPC carcinogenesis is believed to be a multistep process whose progression involved in several the abnormal activities of oncogene and tumor suppressor gene. 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 oncogene in NPC relatively lags behind.Duing 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 oncogene 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 NPC.Genomic alterations can contribute to the dysregulation of expression levels of oncogenes and tumour suppressor genes in cancer cells,the accumulation of which is correlated with tumor progression.Screening for over-expression gene in chromosome amplicon has become the important key to look for candidate oncogene. Results of Redon found that the prevalence of gains or amplifications targeting the long arm of chromosome 3 are common in head and neck squamous cell carcinomason head and neck cancer showed(in＞50%of cases),by combining genome and transcriptional analysis to identify Cyclin L at 3q25.3,especifically amplified and overexpressed in a head and neck cancer cell line,and it is overexpressed consequently to increased gene copy number.Cyclin L,is significantly overexpressed in a population of 20 primary tumors,compared with their normal counterparts,and also found cyclin L overexpression in well-differentiated but not in poorly differentiated primary tumors.Moreover,this gene encodes a putative key regulator of the G0 to G1 transition.Therefore,all these data together lead author to propose cyclin L as a candidate oncogene in head and neck cancer pinpointed as a candidate for a role in promoting cell cycle.With the development of biotechnology,the introduction of genotyping by single nucleotide polymorphism(SNP) arrays has allowed genome-wide, high-resolution analysis of both DNA copy number(DCN) alterations and loss of heterozygosity(LOH) events in cancer cells High-density SNP microarrays permit highly accurate mapping of those genetic changes across the entire genome,providing a promising starting point for the identification of novel candidate genes affected by such genomic abnormalities.Since 2003,the Human Mapping10k,100k,500k SNP Array Set.were introduced by Affymetrix Company.Compared to conventional CGH,karyotype analysis and subsequent array-based CGH,All SNPs on the GeneChip Human Mapping 500K Array Set went through a rigorous screening and validation process. The median physical distance between SNPs is 2.5 kb and the average distance between SNPs is 5.8 kb.The average heterozygosity of these SNPs is 0.30.85%of the human genome is within 10 kb of a SNP.Not only less than 0.02-0.1Mb of chromosome aberrations can be found,but also a lot of sophisticated type of chip-based copy number data analysis software could be employed,such as DNA-Chip Analyzer(dChip) dchip software developed by Stanford University and Affymetrix GTYPE(Ver 4.1) software.All this provide our experimental study with technical support.How the high-density single nucleotide polymorphism(SNP) arrays can detect copy number alterations in cancer? 2004,the investigation of Cheng Li proved that genome-wide analysis of DNA copy-number changes using SNP microarrays could make it entierly possible by three consecutive steps.First,they analyzed 5 cell lines with defined DNA chromosomal copy number(genomic DNA,isolated from cells containing one to five copies of the X chromosome and the constant autosomes),the result of the inferred DNA copy number is consistent with the known karyotape for 99.2%.The inferred copy number calculations based on SNP array hybridization intensity approximate closely to the actual copy number.Next,by analyzing cancer-specific amplification in the NCI-H2171 small cell lung carcinoma cell line by SNP array,and validating each of these amplicate regions by quantitative real-time PCR,the result is also consistent with the known one.Finally,Compared SNP array, cDNA array,and bacterial artificial chromosome(BAC) array log 2 copy number ratios(scatter plots) for BT474 breast carcinoma cell line DNA,they found that the three method detect generally the same types of copy number variation in the same locations.Therefore,SNP arrays have the ability to detect DNA copy number changes.The current researches demostrated that the candidate genes can be detected by integrating genome and transcriptome mapping data,and moreover,this method has a wide range of application in many tumors.2005,the findings of Levi A.Garraway proved that consistent amplified and over-expression critical oncogene,MITF in melanoma-specific was found by employing the above strategy,then they confirmed that the gene played a crucial role in the occurrence and development of melanoma throught expriments.Next,2007,Harada T analyzed the genomic abnormalities characteristic of 27 microdissected PDAC specimens using high-density microarrays representing 116 000 single nucleotide polymorphism(SNP) loci,indicated that frequent gains of 1q,2,3,5,7p,8q,11,14q and 17q(X 78%of cases),and losses of 1p,3p,6,9p,13q,14q,17p and 18q(X 44%) Then,integrating the Ensembl public data,they found that The SKAP2/SCAP2(7p15.2) which belongs to the src family kinases,was most frequently(63%) amplified in sample set and its recurrent overexpression(67%) was confirmed by reverse transcription- PCR.Furthermore, fluorescence in situ hybridization and in situ RNA hybridization analyses for this gene have demonstrated a significant correlation between DNA copy number and mRNA expression level in an independent sample set(P＜0.001).Therefore,they inferred that the dysregulation of SKAP2/SCAP2,which is mostly caused by its increased gene copy number,is likely to be associated with the development of PDAC.Subsequently,the similar analytic methods has been applied in prostate cancer, colorectal cancer,cervical cancer,multiple myeloma,breast cancer,liver cancer and other tumor cell lines.Previously,Ph.D Zhongxi Huang constructed more tree model for NPC carcinogenesis using 170 comparative genomic hybridization(CGH) samples data from five smaller studies.The tree model suggested that the amplification region within 12p12-11 may also be an early event during NPC carcinogenesis.And moreover,over-amplified region of chromosome arm 12p in other tumors is an high-frequency amplification event.Based on the previous study,we inferred that the high frequence amplicication region 12p12-11 may contain critical candidate oncogenes of NPC.Therefore,to screen for both amplified and overexpression gene within amplicon 12p12-11 in NPC, 250k SNP array and expression microarray were used to assay five NPC cell lines and NP69 simultaneously.Furthermore,fluorescence quantitative RT-PCR and immunohistochemical methods were employed to evaluate the candidate oncogenes in NPC preliminarily.The identification of its characteristics in NPC tissues and cells will help us understand that the candidate gene contributes to tumorigenesis of NPC.1.Integrated genomic and transcriptomic mapping data identifies candidate oncogenes in NPCFive NPC cell lines(HONE1,CNE1,CNE2,5-8F,and 6-10B) and an immortalized nasopharyngeal epithelial cell line NP69 were evaluated with 250K SNP arrays(Affymetrix) to detect the genome-wide DNA copy number alterations. Simultaneously,we did expression microarray analysis on three NPC cell lines (HONE1,CNE1,and CNE2) and NP69 with Affymetrix Human Genome U133 Plus 2.0 microarray containing 54,675 probe sets.NP69 was used as normal control. PTHLH gene located in the region of 12p12-11 which was predicted as early event in NPC by tree model previously(but not in the CNV regions) was found to be gained and over-expressed in NPC by our method.2.Identification of the copy number change and expression characteristics of PTHLH in NPC tissues and cell linesQRT-PCR was used to verify the DCN and expression of PTHLH gene in the six cell lines.The results of one-way ANOVA ananlysis showed that the copy number change of PTHLH in six cell lines and placenta tissue was significantly different from each other(P＜0.05).the results of an absolute quantitative method were indicated that the copy number of PTHLH was significantly exceeded in six cell lines as compared with placenta tissues.The results of 2^{-△△Ct} method showed the expression of PTHLH was significantly increased in five NPC cell lines as compared with NP69(P＜0.05).It was interesting that the expression of PTHLH gene in 6-10B and CNE1 was higher than in 5-8F and CNE2.Overexpression of PTHLH in relatively large samples of NPC tissue was further confirmed by RT-PCR,the results showed that PTHLH was expressed in 64.7% (11/17) NPC tissues.Immunohistochemistry was used to detect the expression of PTHLH in in 61 cases of paraffin slides(including 49 NPC and 12 NP).The results showed that PTHLH gene mainly located in cellular membrane and cytoplasm in NPC cases.PTHLH can also be found to be located in nucleus in a few cases.In addition,PTHLH was expressed in 91.8%(45/49) NPC tissues,which was much higher than in NP.The difference between two groups was statistically significant (P＜0.05,Mann-Whitney U test).No correlation of PTHLH expression was found between sex and age(P＞0.05).Conclusions1.This method that the cancer samples are simultaneously evaluated by genome-wide SNP arrays and expression microarrays can identify candidate oncogene in cancer.PTHLH,the candidate oncogene is detected in NPC cell line within chromosome high level amplification 12p12-11 region.and also high level amplification of 5p15.33 region was another novel finding of the present study.2.PTHLH is verified consensual amplification and overexpression in NPC cell lines by real-time PCR.The results of RT-PCR and Immunohistochemistry showed that PTHLH was expressed in NPC tissues significantly higher than NP. All this analysis predicts PTHLH is an candidate oncogene of NPC,might be involved in origin and progression of NPC.