Exploring The Molecular Mechanisms Of Tumorigenesis And Metastasis In Nasopharyngeal Carcinoma Cell Lines 5-8F And 6-10B

BACKGROUND & OBJECTIVENasopharyngeal carcinoma (NPC) is a special kind of malignat head and neck cancer with high incidence in Southeast Asia and Southern China. Tumor formation and metastasis is a complex and continuous process that needs multi-step, multi-stage, multi-factor involving in a number of key oncogenes and tumor suppressors. But its molecular mechanism is not very clear. So far, Epstein-Barr virus (EBV), certain chemical carcinogens and genetic factors are thought to be closely associated with the pathogenesis of NPC.Oncogenesis and metastasis of tumour is the most difficulties during the clinic therapy. It also has attracted keen interest and further study of many tumour basic researchers.Then, which genes may affect the tumour genesis? Which genes and its pathways may play a vital role in the course of NPC metastasis? The past studies have found some oncogenes, tumor suppressors, metastasis promoted genes and metastasis suppressed genes. However, there were lots of key genes that have not been found up to now. So we will continue to search the potential oncogenes by way of bioinformatics and do experimental verification. At the same time, we will also to excavate the metastasis associated genes of NPC and the pathways.During the early stage research of our team, by integrating genomic and transcriptomic mapping data, PTHLH gene located in the region of 12p12-11 which was predicted as early event in NPC by tree model previously was found to be gained and over-expressed in NPC cell lines. QRT-PCRwas used to verify the DCN and expression of PTHLH gene in the NPC cell lines. The result was nearly in accord with the array data. Overexpression of PTHLH in relatively large samples of NPC tissue was confirmed by RT-PCR, the results showed that PTHLH was expressed in-64.7% NPC tissues.Immunohistochemistry was used to detect the expression of PTHLH in 61 cases of paraffin slides (including 49 NPC and 12 NP).The results showed that PTHLH was expressed in NPC tissues significantly higher than NP. All this analysis predicts PTHLH is a candidate oncogene of NPC, might be involved in origin and progression of NPC.So in the part one of this topic, molecular biologic experiment will be used to validate the tumour formation capability of PTHLH in NPC cell line.Metastasisi of NPC is the main reason for the failure of treatment. However people know little about its molecular mechanism. Therefor, further selection and identification of the genes crucial for the metastasis of NPC is of great significance, not only for basic understanding of the molecular and cellular processes involved in NPC metastasis, but also for providing new potential therapeutic targets.Both of the 5-8F and 6-1 OB cell lines are originated from the SUNE-1 NPCcell lines. Previous work has shown that the 5-8F cells are highly tumorigenic and metastatic while 6-10B cells just tumorigenic and less-metastatic. These two cell lines have been used successfully in several differential transcriptomic/proteomic analyses. But the previous high-throughput technologies can only covered less than 8000 of approximately 25,000 human genes. Considering the limited throughput of the previous transcriptomic/proteomic assays, in the part two of this topic, we thought to use a high-throughput microarray-based approach to more comprehensively study the genetic signatures of 5-8F and 6-10B cell lines, find the differentially expressed genes and the pathways between the two cell lines.METHODS1.Expression characterization of PTHLH in NPCTo study the differential expression levels of PTHLH between NPC cell lines, fluorescence quantitative PCR and western blot methods were used to detect the PTHLH expression in immortalized nasopharyngeal epithelial cell NP69 and NPC cells 5-8F,6-10B, CNE1,CNE2 and HONE1 in the mRNA and protein levels. Combination of the array data, the cell line that showed a high mRNA and protein expression level of PTHLH was selected for the functional validation.2. The influences on cell biology of the NPC cells by knock-down of PTHLHConstructing PTHLH interference and blank lentivirus expression vectors to infect 6-10B cells.The cell clones with stably expression of anti-PTHLH shRNA and blank vectors were picked out. Fluorescence quantitative PCR was used to detect the interfere efficiency of each cell clone. The cell clone with highest interference efficiency-would beselected as the next experiment object.MIT, flow cytometry and colony forming experiments were used to detect cells proliferation in vitro;Transwell was employed to detect cells migration in vitro;Then, after the target gene was successfully silenced, the Fluorescence quantitative PCR method was used to detect the change of expression levels of the genes that may interact with PTHLH.3. Microarray analysis of differentially expressed genes between nasopharyn-geal carcinoma cell lines 5-8F and 6-10BThe mRNA samples of 5-8F and 6-10B cells were provided by us to the company. Three biologic replicates of each were run on the Affymetrix Human Genome U133 Plus 2.0 microarray containing 54,675 probe sets. Two recently published software suites were used to explore the differentially expressed genes and to identify the molecular mechanisms associated with the different biologic characteristics of these two cell lines. Two specific tools were used:GOEAST (Gene Ontology Enrichment Analysis Software Toolkit;http://www.omicslab.genetics.ac.cn /GOEAST/) and GenCLiP (a software program for clustering gene lists by literature profiling and constructing gene co-occurrence networks related to custom keywords; http://www.genclip.com). We intended to find the candidate genes that are closely related with the metastasis, and do the experimental validation.4. Statistical methodsWe use SPSS 13.0 statistical software package for statistical treatment,QRT-PCR detection results were analyzed using one-way ANOVA, and multiple comparison test using SNK. In Chapterâ…¡, QRT-PCR,migrate, flat colony formation were tested by one-way ANOVA, MTT results using factorial design analysis of variance, multiple comparisons test using SNK or LSD test.RESULES1. Expression characterization of PTHLH in NPC1)The results of fluorescence quantitative PCR showed PTHLH expression with statistical significance(F=100.473,P=0.000)in immortalized nasopharyngeal epithelial cell line NP69 and NPC cell lines 5-8F,6-10B, CNE1,CNE2, and HONE1.HONE1 cell line showed the highest expression of PTHLH in all 6 NPC cell lines.The expression of PTHLH in 6-10B was higher than 5-8F (5 fold).2) Western blot was used to detect the protein level of PTHLH in NPC cell lines 5-8F,6-10B,CNE1,CNE2 and HONE1.And the Image Analysis Software "Image-Pro Plus" was used to calculate the OD optical density of each band, thereby we could know the expression of PTHLH in all of the NPC cell lines. The result was that PTHLH protein was high expressed in 6-10B,CNE2 and HONE1 cell lines, meanwhile the 6-10B and HONE1 showed the highest expression. Combination of the array data, we chosed 6-10B cells as the interference object for further experiment study.2. The influences on cell biology of the NPC cell by knock-down of PTHLH1) The lentiviral vectors containing specific anti-PTHLH shRNA and negative control were transfected into 293FT cell line to produce lentivirus particles, followed by transduced into 6-10B cell line. After selected by limiting dilution analysis for 14 days,4 monoclones which were GFP-positive were picked out, fluorescence quantitative PCR was used to detect the interfere efficiency for each clone. Clone2 with the highest efficiency (94.1%) was chosen for the following study. There is markedly differentiation between positive clone and negative control clone,6-10B in PTHLH expression (F=59.833, P=0.000).Interference clone was named 6-10B/PLVTHM-PTHLH, negative control clone named 6-lOB/PLVTHM.2) A significantly time-dependent inhibitory proliferation was found in 6-10B/ PLVTHM-PTHLH cells compared with 6-lOB/PLVTHM and 6-10B by in vitro MTT assay (F=65526.028,P=0.000), Flow cytometry results showed no obvious differentiation among the-three cells in S phase and G2/M phase change (G1 P=0.132,S P=0.076,G2/M P=0.109). In addition,6-lOB/PLVTHM-PTHLH cells had a significant impaired ability to form colonies in plates, as compared with 6-lOB/PLVTHM and 6-10B cells(F=118.940,P=0.000).The results indicate that silence of PTHLH suppressed proliferation of 6-10B cells.3) 6-lOB/PLVTHM-PTHLH cells had a significantly reduced ability of migration as compared with 6-10B/PLVTHM and 6-10B cells detected by Transwell assay in vitro (F=1966.153,P=0.000). The result indicated that silence of PTHLH reduced migration of 6-10B cells. 4) Fluorescence quantitative PCR was used to detect the variation of expression level of DKK1 and b-catenin in the positive clone. The result reveal that relative to 6-1 OB and negative control cell, the expression level of DKK1 and b-catenin had nearly no variation. The difference had no statistical significance (F=0.104 P=0.903/F=0.210 P=0.816).3. Microarray analysis of differentially expressed genes between nasophary-ngeal carcinoma cell lines 5-8F and 6-10BBy microarray analysis, only 60 differently expressed genes were identified between the two cell lines.To validate the differently expressed genes identified by expression microarray analysis, we performed qRT-PCR for 8/60 genes. The qRT-PCR results for each of the eight genes analyzed showed high concordance with normalized log2 ratio values derived from expression microarray analysis. Gene ontology analysis showed that most of these genes participated in cellular and metabolic processes, and the primary molecular functions of each were catalytic activity, ion binding, and protein binding. GenCLiP analysis showed that these genes were specifically related to apoptosis, cell cycle,metastasis, chemokines, and immunoediting, but not cancer, NPC, stem cells, lymphangiogenesis, angiogenesis, inflammation, nor proliferation.In particular, 42/60 genes have established metastatic functions (P<0.00001), while 11 out of those 42 genes formed gene networks related to metastasis (P= 0.013). Among the genebnetworks identified, the PTHLH gene was of particular interest. Predicted to regulate the WNT pathway through the DKK1 gene, the PTHLH gene may affect tumorigenesis and metastasis of NPC and merits further study.CONCLUSION1) The results of fluorescence quantitative PCR and western blot showed that the expression of PTHLH were different among the NPC cell lines 5-8F,6-10B, CNE1,CNE2 and HONE1.The 6-10B and HONE1 cell lines showed the highest expression of PTHLH. We chosed 6-10B cell line for RNAi of the PTHLH gene.2) The down-regulation of PTHLH inhibited the proliferation and migration of the 6-10B cell line. It incated that PTHLH can improve the tumor generation of 6-10B cell line.3) There were 60 differentially expressed genes between 5-8F and 6-10B cell lines found by microarray analysis. Bioinformatic analysis showed that most of these genes participated in cellular and metabolic processes, and were specifically related to apoptosis, cell cycle,metastasis,chemokines, and_immunoediting. Among these genes, the DKK1 gene was predicted to affect tumorigenesis and metastasis of NPC through the WNT pathway. But the activity of DKK1 was not affected by the expression level of PTHLH. PTHLH was an oncogene in NPC, but it was not associated with the metastasis of NPC.