The Screening And Functional Study Of Amplified Genes Located At Chromosome 12p12-11 In Nasopharyngeal Carcinoma

Nasopharyngeal carcinoma (NPC) is one of the malignant tumors with a high incidence and causing a serious health problem in Southeast Asia and Southern China, which has distinct geographical and ethnic distribution. More than 95% of NPC is poorly differentiated and undifferentiated with high incidence of early metastasis (metastasis is the main cause of death in NPC). Radiation therapy is currently the first choice for NPC treatment. Although the radiotherapy equipment and technology were improved tremendously, but the five-year survival rate of NPC did not radically change and remains at about 50%-60%. Therefore, it is of importance to explore its causes and find a new and effective treatment to NPC.Studies on epidemiology and etiology of NPC have shown that genetic factors (genetic susceptibility), EB virus infection and other environmental factors (mainly chemical carcinogens, etc.) are related to the occurrence and development of NPC. Under the combined effect of these factors, the genetic variation events affecting normal nasopharyngeal epithelial cells and the continuous accumulation of epigenetic changes led to activation of oncogene and inactivation of tumor suppressor genes, ultimately resulting in malignant cellular transformation. The current studies show that significant ethnic susceptibility to NPC and familial aggregation phenomenon indicate that changes of genetic genes play an important role in NPC, but compared to other human common malignant tumor, there is little known facts about the basis of molecular pathogenesis of NPC and the associated change in molecular genetics. The specific molecular mechanisms of NPC incidence have not been elucidated yet, so it is of great significance to look for and identify the genes closely related to the development of NPC and study their functions which is the inevitable way to reveal the molecular mechanism of NPC. Past research found that some proto-oncogenes(for example, HRAS, KRAS2,cyclin D1, MDM2, STAT3, EGFR, etc.) and tumor suppressor genes (P53,P16,RASSF1A, DLC-1, LTF, BLU, etc.) were expressed abnormal in NPC, but not yet confirmed as nasopharyngeal cancer-specific tumor gene or tumor suppressor gene. Therefore, we need to understand and access the information of the significant oncogenes and tumor suppressor genes related to NPC from a new perspective angle.Zhi-Hua Yin and Zhong-Xi Huang in our Lab have constructed a tree model of NPC tumorigenesis to explore the pathogenetic mechanism of NPC by using comparative genomic hybridization data of 170 cases of NPC from five laboratories in Hong Kong, Taiwan, Hunan and Guangdong. The study showed 11 chromosomal aberrations in NPC, among which 2 early events in the pathogenesis of NPC were-3p26-13 and+12p12, the latter is the first reported. LOH and chromatid transfer experiments suggested the loss of 3p is an early event in NPC as well as some NPC tumor suppressor genes might exist at this region. Recent research further indicates that there are several candidate tumor suppressor genes (such as RASSF1A, BLU and LTF) located at 3p21.3, and also there are hypermethylation of gene promoter regions, leading to down regulation of gene expression. The evidences mentioned above suggest that the tree model prediction is accurate. Therefore, the amplification based+12p12-11 as the early event of NPC provides a new entry point to find the key oncogenes in the pathogenesis of NPC.[The screening of amplified genes located at 12p12-11]Chromosomal gain often leads to the amplification in copy number and overexpression of oncogene and it's important to search for specific tumor-related genes by detecting over-expression of candidate oncogenes in the amplified chromosome region. Chromosome 12p12-11,19Mb at length, contains 123 known genes and expressed sequence tag (expressed sequence tag, EST). Previous studies have shown that,+12p is an early event in colorectal cancer, and that may be the late events of germ cell tumors or late events of ovarian cancer. We preliminarily determined 18 candidate tumor-related genes, among which 6 real oncogenes were determined(DDX11, LDHB, CASC1, KRAS2, BCAT1, PTHLH) and other 12 genes (SOX5, PDE3A, CMAS, BHLHB3F, KCNJ8, PKP2, EKI1, PTX1, DNM1L, SSPN, RECQL, SLCO1A2) were associated with cell transformation and proliferation by using MEDLINE database and NPC cDNA microarray data from our laboratory.We determined these 18 gene expression level between 36 cases of NPC and 8 cases of chronic nasopharyngitis tissues by RT-PCR and quantitative RT-PCR. The results showed 9 genes (RECQL, PDE3A, SLCO1A2, ASC1, DDX1, SSPN, PTHLH, BHLHB with PKP2) not expressed in NPC and in chronic nasopharyngitis tissues and the expression of 5 genes (CMAS1, DNM1L, ETNK1, LDHB and SOX) has no significant difference between 36 cases NPC and 8 cases chronic nasopharyngitis tissues (P> 0.05); While 4 genes (BACT1, KCNJ8, PTX1 and KRAS2) in NPC tissues were expressed significantly higher than that in controls (P<0.05), suggesting these 4 genes may be relevant to the development of NPC.KCNJ8 is categorized into a sub-class of the ATP-sensitive potassium ion channel gene in relation to cell secretion and muscle contraction. At present there is no evidence that it is associated with the occurrence of cancer; PTX1 mainly involved in membrane transport of materials in endoplasmic reticulum and Golgi apparatus, down-expressed only found in prostate cancer; there are more studies of KRAS2 in NPC, its expression was significantly higher than that of the normal nasopharyngeal tissue and nasopharyngitis. BCAT1 (branched chain aminotransferase 1 gene) in Burkitt's lymphoma and breast cancer tissues were expressed significantly in relation to cell proliferation and apoptosis, but there is no relevant reports in NPC. Therefore, we select BCAT1 as a target for further study to explore its relationship with NPC development.[the expression of BCAT1 protein in different NPC pathological stages]To study tumor-related oncogenes selected from the 12p12-11 in various stages of precancerous lesions can be aware of the significance of these genes in NPC pathogenesis. Therefore, we used immunohistochemical method to analyze the expression of BCAT1 protein in the normal nasopharyngeal epithelium, low-moderate grade pre-invasive lesion, high grade pre-invasive lesion and NPC. The results showed that in the normal psudo-stratified ciliated epithelium, stratified epithelium, low-moderate grade pre-invasive lesion, high grade pre-invasive lesion and NPC, different levels of protein expression, positive signal located in the cytoplasm were able to be detected; The positive rate of the expression of BCAT1 protein in normal epithelium, low to moderate grade pre-invasive lesion, high grade pre-invasive and NPC were 23.6%(17/72),75%(18/24),88%(8/9) and 88.75 (71/80) respectively. The results suggest the expression of BCAT1 significantly increased (P<0.05) with the hyperplasia occurring in the nasopharynx of early pathological changes, which indicates the BCAT1 may play an important role in the occurrence of NPC and also provide a basis for further analysis of the molecular mechanism of gene expression abnormality.[Explore possible mechanisms about abnormal expression of BCAT1 in NPC] Genetic alterations and transcription des-regulation are two approaches to explore the mechanism of tumor gene up-regulation. Firstly, we analyzed the mutation status of 11 exons of BCAT1 from 20 cases of NPC by PCR-sequencing and one SNP site in exon1 was detected, suggesting mutation had no significant effect on the expression of BCAT1. Secondly, the copy number amplification is considered as a mechanism to activate an oncogene. Analyzing the amplification of microsatellite loci genes within and near the gene can indirectly reflect the gene amplification in the genome. We analyzed the amplification of three microsatellite loci (D12S1435, D12S1617 and RH44650) within BCAT1 by Real-time PCR in NPC. The results showed that 42.4%(12/28) of the NPC tissue have the amplification of microsatellite loci in BCATl. All above suggested that increase of the copy number may play a role in BCAT1 upregulation in NPC tissues to some degree.Regulation of eukaryotic gene transcription is a very complicated process, and the participation of transcription factors play an important role in regulation of gene transcription. Bioinformatics analysis shows that BCAT1 5'untranslated region-155/-200bp area contains a c-Myc binding sites. In order to reveal whether c-Myc has a direct role in regulation of the expression of BCATl, we carried out chromatin immunoprecipitation experiments (ChIP). The results confirm c-Myc transcription factor can be directly binding the c-Myc binding site in BCAT1 gene promoter region. Further, we used RNAi to establish a stable 5-8F NPC cell line (named 5-8F/Si-c-Myc) with c-Myc expression interference and found that blocking of endogenous c-Myc expression could reduce the mRNA expression level of BCATl. In addition, we established a recombinant reporter gene vector by connecting BCAT1 regulatory sequences including c-Myc binding sites with the luciferase reporter gene, and then transfected 5-8F cell,5-8F cell with the blank vector (5-8F/si-control), as well as 5-8F/si-c-Myc cell. The reporter gene activity analysis showed that reporter gene activity of recombinant luciferase decreased after knockdown of endogenous c-Myc expression compared with the control group. The experimental results show that c-Myc has a direct positive regulatory role in the expression of BCAT1.[Study the function of BCAT1 in 5-8F by RNA interference technology]We constructed RNAi vectors targeting BCAT1 (shBCAT1) by pSUPER.retro retrovirus vector system. After the results confirmed by sequencing, we transfected shBCAT1 into 5-8F using the lipofection technique and puromycin resistant clones were selected, PCR confirmed that resistant clones contain shBCAT1; RT-PCR results showed that shBCAT1 expression vector could specifically lead to expression of BCAT1 reduced by 90%. Western Blotting analysis showed that the protein expression of BCAT1 decreased 55% or so in 5-8F. These results confirm that we have successfully established a stable interference system for BCAT1 gene in 5-8F.Subsequently, we further examined the effects on 5-8F biological characteristics after the BCAT1 was blocked. Flow cytometric analysis showed that cell cycle distribution of 5-8F and 5-8F-blank vector did not change significantly; The 5-8F-shBCAT1 caws blocked in the G1 phase, G1 phase cells increased～16.66%(16.66±2.92,P=0.005), while the G2-phase cells decreased by～6.91%(6.91±0.76. P= 0.001). MTT assay found that the cell proliferation of 5-8F-shBCAT1 rate was significantly lower than control group,5-8F and 5-8F-blank vector, and the difference was statistically significant (P= 0.00). In addition, migration assay and invasion assay demonstrated that 5-8F-shBCAT1 could also significantly decrease the mobility and invasion ability of 5-8F cells.[The interaction between BCAT1 and LARS2 and the inactivation Mechanism of LARS2 in NPC]Bioinformatics is a tool for post-genomic era. STRING (http://string80.embl.de/) is a known and widely used database which reveals the protein-protein interaction between the known and predicted protein. The protein-protein interaction includes both the direct (binding) and the indirect interaction(association) and the background data come from genomic analysis, high-throughput experiments, co-expression experiments and published databases (such as the PUBMED, MIPS). At the same time, the data among different species can be compared by STRING. The latest version STRING 8.1 contains 630 species, more than 2,590,259 proteins. We found 10 protein molecules directly and indirectly interacted with BCAT1, which are LRMP, hCG₂6005, ENSP00000372, BCKDHA, BCKDHB, IARS2, IARS, LARS2 and LARS. Moreover, we found that LARS2 gene is located in 3p21, which has a high-frequency loss in NPC. LARS2 (leucyl aminoacyl-tRNA synthetase 2) gene encodes leucyl-tRNA synthetase. In order to explore the possible role of LARS2 in NPC, we used RT-PCR and Real-time PCR to detect the expression of LARS2 in 8 cases of chronic nasopharyngitis and 36 cases of NPC. The results showed that, the mRNA expression of LARS2 was down-regulated or deleted in 78%(28/36) of the NPC tissue, suggesting that LARS2 may be a candidate tumor suppressor gene in NPC. Then, we studied the primary mechanism for LARS2 inactivation from the aspects of genetics and epigenetics. Firstly, we studied mutation in the promoter region and exon 1 in 25 cases of NPC by PCR-SSCP combined with sequencing analysis. The results did not detect the mutation and SNP, indicated that mutation had no effect on inactivation of LARS2 expression. Secondly, as the loss of microsatellite loci within and near the gene can indirectly reflect the absence of genes in the genome, we used PCR-denaturing polyacrylamide gel electrophoresis to analyze the loss of the two microsatellite loci (RH25266 and SHGC-12886) within LARS2 in NPC. The results showed that at least 28%(7/25) of NPC have loss of microsatellite loci in LARS2, suggesting that LOH of microsatellite loci may downregulate the expression of LARS2 in NPC in a certain degree. Thirdly, hypermethylation of gene promoter is the most common epigenetic change of tumor suppressor gene. Therefore, whether there exist CpG islands in the promoter region of LARS2 was analyzed and a 227 bp long CpG island in the promoter region was found. Then, we designed the methylation and the non-methylation-specific primers of this region. Finally, we analyzed methylation status of LARS2 promoter in the chronic nasopharyngitis and NPC by methylation-specific PCR (methylation-specific PCR, MSPCR) technology; The results showed that 64%(16/25) of NPC tissue and 12.5%(1/8) of Chronic nasopharyngitis tissue have the LARS2 gene promoter methylation, but the methylation ratio of LARS2 in NPC is significantly higher than that in chronic nasopharyngitis tissue. Statistical significant difference exists between the two groups (P=0.017), suggesting that promoter methylation is the main factor leading to down-regulation of LARS2.Further comparison showed that both BCAT1 and LARS2 proteins participated in the same cell metabolism pathway, namely, leucine metabolism. Up-regulation of BCAT1 and down-regulation of LARS2 in NPC together led to accumulation of the intermediate product-L-leucine. As a branched-chain amino acid, L-leucine activates mTOR signaling pathways by changing the mTOR (mammalian target of rapamycin)-raptor complex stability. Studies have shown that mTOR is often highly expressed in NPC. On one hand, high expression of mTOR lead to uncontrolled proliferation of malignant cells eventually by the downstream S6K1 and 4E-BP1 molecules regulating mRNA translation of cell cycle required protein such as G1 phase and S phase protein [such as cyclin D1, Rb protein, hypoxia-inducible factor-1 (hypoxia inducible factor-1, HIF-1), c-Myc and vascular endothelial growth factor (vascular endothelial growth factor, VEGF), CLIP-170, etc.]. On the other hand, high expression of mTOR can also affect epithelial cell differentiation by inducing the expression of DNA transcription factor Id2. Based on the above experiments and analysis, we believe that there is an indirect interaction relationship between the up-expression of BCAT1 and down-expression of LARS2, which together involved in promoting the occurrence and development of NPC.In summary, through this study, we found a NPC-related oncogene-BCAT1 from the early event of NPC located at+12p12-11 for the first time, and systematically and comprehensively examined the expression level of BCAT1 protein in chronic nasopharyngitis, NPC and nasopharyngeal cancer tissue at different stages and studied the possible mechanisms of upregulation of it in NPC. The results suggest BCAT1 protein has normal expression in chronic nasopharyngeal epithelial tissue while the expression significantly increased in NPC and different stages of nasopharyngeal carcinogenesis. The upregulation was mainly due to gene amplification, while the c-Myc also played a role for its direct regulation of BCAT1. RNAi showed that the BCAT1 knockdown could inhibit the proliferation of 5-8F, leading to cell cycle arrest. In addition, we discovered that LARS2 protein could interact with BCAT1 protein indirectly by bioinformatics studies. LARS2 is significantly down-regulated in NPC and promoter hypermethylation is the main mechanism of its inactivation. Up-regulation of BCAT1 and down-regulation of LARS2 in NPC together led to accumulation of the intermediate product-L-leucine, which could lead to uncontrolled proliferation of malignant cell by activating mTOR signaling pathway. The bioinformatics predicting results remain to be elucidated by experimental validation.