| Oral Malignant tumor is a disease threatening human’s health.90% of oral cancer is oral squamous carcinoma.Like the occurrence of other tumors, the occurrence and development of OSCC, is a long and complex process coefficient by multi-step and multi-stage of poly-molecule matter with network structure. And it’s also a consequence coefficient by poly-genes. Inter-synergy of all oncogene formulates and develops the tumor cell.Now there is no major breakthrough for the occurrence mechanism and cure of tumor. Early detection and radical cure of tumor is a puzzle and chance to the presentThe solution to this puzzle is a result of overall analysis to the occurrence and development of tumor. Researchers have made intensive study and made some progress. But the why tumors occur is still indefinite. What is universally accepted is that tumor is a disease of molecular level, and tumor research from protein level of gene is the ultimate way.In recent years, genomics based on scanning of high throughput molecule, proteomics and the technical integration of CAD and the application of correlative technical chains provide turning points to the research of tumor and make abundant achievement in the research of breast cancer, lung cancer, gastric cancer, colon cancer, ovarian cancer and melanin tumor.RACK 1,as a node molecule, attracting great attention of many scholars is protein kinase receptor protein, which can interact with extensive kinase and receptor and mediate the activity of much cytobiology. There is a popular belief among scholars that some important biologic reaction such as cell multiplication, transferring, and invasion may pay a decisive regulating effect. In abundant research, relation between RACK1 and neoplasia and transferring is a hotspot in recent-year’s tumor research.Gene microarray is the most reliable and widely-used bioclip, called DNA clip, DNA microarray. As for how to make it, plenty of oligonucleotides or Edna fragment should be designed in advance and arranged in carriers in high density and in good serial to make clip.Samples to be tested should be marked in fluorescent dye(such as Cy3, Cy5) and be prepared as probe, which is to be cross-fertilized with clip on principles of base complementary pairing. Through laser focus and gathering hybridization signal through fluorescence scanning, serial information about sample molecular can be gained. Through computer analysis image and data can be obtained.In this way, timing analysis and research of gene serial and function can be carried out in the whole genome and large numbers of data can be obtained at a time for parallel analysis. Doing like this can both accelerate the speed of experiment and improve the accuracy of analysis. Analysis of several objective gene on one clip can eliminate inherent difference resulted from many factors, then large-scale, high-throughput, high-sensitivity and high-accuracy test research can be conducted.With gene expression profile clip, this test is to detect oral squamous cell carcinoma and other organization by the side of oral squamous cell carcinoma. Differential gene with clear change can be screened out from the result of gene expression profile clip and bioinformatics analysis can be carried out. By referring to earlier IPA analysis and test result proteomics, how to interact among the differential genes and 52 related differential protein is clear and gene different from OSCC can be screened out. verification for RT-PCR and establishment of mutual function of relational network will be ultimately done. Establish the network of these differential genes’interactions by STRING database. And analysis the function,3-dimension structures,structure regions and the phosphorylation position of the node genes through NCBI, NEXTPROT, EMBL-EBI, NetPhos2.0 databases, following which the farther confirmatory experiments would be done to make sure the mechanisms of these differential genes.Chapter 1 Inspection of Gene Expression microarray of Oral Squamous Cell CarcinomaPurpose:With gene expression profile clip, this test is to detect oral squamous cell carcinoma and other organization by the side of oral squamous cell carcinoma. Differential gene with clear change can be screened out from the result of gene expression profile clipMethod:gathering 2 excision of oral squamous cell carcinoma and 2 excision of normal structure beside it of Stomatological Hospital of Guangdong Province in 2013, and detecting oral squamous cell carcinoma and normal structure beside it with the help of NimbleGen Human Gene Expression Microarrays(Microarrays version:Roche NimbleGen Homo_ Sapiens 12x135k)Result:according to screening criteria of differential gene,7872 out of 32448 detected gene are differential expression gene of oral squamous cell carcinoma, which accounts for 24%.3800 are up-regulation expression gene, and 4072 are down-regulation gene.Conclusion:through detection with the help of gene expression profile clip, 7872 differential expression gene are obtained according to the criteria that differential expression doubles. Thus it can be concluded that the occurrence and development is not a result of single or several gene. The anterior experiment based on single or several gene is local. It can also be concluded that the occurrence of tumor is a result of mutual regulation effect of many gene forming a network, which is quite complicated.Chapter 2 Bioinformatics analysis of gene expression microarrays dataPurpose:We compared the gene expression differential of the oral squamous cell carcinoma and the tissue by the side of oral squamous cell carcinoma through gene expression microarrays.To contact the data of gene expression microarrays and the biological function,we should to do the functional clustering analysis of the gene data.In this part of the experiment we did the Bioinformatics analysis about the test results of the gene expression microarrays to find the variation trend and the interaction net work of the differential gene chosen.Method:We analysed the data of the gene expression microarrays in the first part by the software of Agilent GeneSpring GX v12.0 supported by the gene microarrays.Result: We did the GO analysis and the KEGG pathways analysis by the the software of Agilent GeneSpring GX v12.0 supported by the gene microarrays,which depart the result by up regulation and down regulation of the gene.The first, the distribution of the differential gene between the oral squamous cell carcinoma and the tissue by the side of carcinoma by the method of the GO analysis:1 Biological Process1.1 The up regulation gene in the biological process part:532 subhorizon of Biological Process were found by the functional enrichment analysis of the up regulation differential gene,which are mainly enrichment in:response to stimulus (6.75), immune system process (6.06), extracellular matrix organization (5.54), regulation of immune system process (5.49), immune response (5.39), organ morphogenesis (4.88), extracellular structure organization (4.84), lymphocyte costimulation (4.77), T cell costimulation(4.77), positive regulation of cell activation (4.75)1.2 The down regulation gene in the biological process part:715 subhorizon of Biological Process were found by the functional enrichment analysis of the down regulation differential gene,which are mainly enrichment in:signaling(9,70), biological regulation(9.29), death(9.12), cell death(8.98), cellular process(8.89), signal transduction(8.74), cellular component movement(8.69), response to wounding(8.24), regulation of biological quality (8.11), wound healing(7.90).2 Cellular Component2.1 The up regulation gene in the Cellular Component part:41 subhorizon of Cellular Component were found by the functional enrichment analysis of the up regulation differential gene,which are mainly enrichment in:extracellular region part(12.14),extracellular region(12.14), extracellular space(10.91). plasma membrane part(7.69), integral to plasma membrane(7.50), intrinsic to plasma membrane(7.49), extracellular matrix(6.00), fibrillar collagen(5.53), plasma membrane(5.02), cell periphery(4.93)。2.2 The down regulation gene in the Cellular Component part:149 subhorizon of Cellular Component were found by the functional enrichment analysis of the down regulation differential gene,which are mainly enrichment in:cytoplasm (18.17), cytoplasmic part (11.18), intracellular part (9.07), plasma membrane part (9.01), intracellular (8.88), cell fraction (8.79), cytosol (8.77), insoluble fraction (8.65), membrane fraction (7.99), cell periphery (7.31)3.Molecular Function part3.1 The up regulation gene in the Molecular Function part:97 subhorizon of Molecular Function were found by the functional enrichment analysis of the up regulation differential gene,which are mainly enrichment in:signal transducer activity(6.74), molecular transducer activity(6.74), receptor activity(5.50), extracellular matrix structural constituent(5.30), carbohydrate binding(5.07), heme binding(3.71), transmembrane receptor activity(3.58), sodium ion transmembrane transporter activity(3.53), sugar binding(3.44), heparin binding(3.42)。3.2The down regulation gene in the Molecular Function part: 132 subhorizon of Molecular Function were found by the functional enrichment analysis of the down regulation differential gene,which are mainly enrichment in:protein binding (17.68), enzyme binding (8.97), cytoskeletal protein binding (8.00), binding (7.69), actin binding (6.30), binding, bridging (5.43), protein heterodimerization activity (4.96), protein dimerization activity (4.65), kinase binding (4.64), SH3 domain binding (4.46)。The second The data of the gene expression microarrays between the oral squamous cell carcinoma and the tissue by the side of carcinoma by the analysis of the KEGG pathway enrichment:We did the KEGG pathway analysis of the differential expression gene data according to the up-to-date KEGG database.The biological pathway of the differential expression gene enriched in:1.1 The biological pathway of the up regulation differential gene.The biological pathway of the up regulation differential gene were found in 26 biological pathways,which are mainly enrichment in:Staphylococcus aureus infection-Homo sapiens (human) (4.59), Neuroactive ligand-receptor interaction-Homo sapiens (human) (4.46), Asthma-Homo sapiens (human) (4.25), DNA replication-Homo sapiens (human) (3.80), Intestinal immune network for IgA production-Homo sapiens (human) (3.28), Graft-versus-host disease-Homo sapiens (human) (2.84), Salivary secretion-Homo sapiens (human) (2.74), Cytokine-cytokine receptor interaction-Homo sapiens (human) (2.72), Systemic lupus erythematosus-Homo sapiens (human) (2.52), Metabolism of xenobiotics by cytochrome P450-Homo sapiens (human) (2.19), Antigen processing and presentation-Homo sapiens (human) (2.19)。1.2 The biological pathway of the down regulation differential gene.The biological pathway of the down regulation differential gene were found in 55 biological pathways,which are mainly enrichment in:Amino sugar and nucleotide sugar metabolism-Homo sapiens (human)(6.19), Epithelial cell signaling in Helicobacter pylori infection-Homo sapiens (human)(6.14), NOD-like receptor signaling pathway-Homo sapiens (human)(4.88), Pathways in cancer-Homo sapiens (human)(4.52), Adherens junction-Homo sapiens (human)(3.93), Shigellosis-Homo sapiens (human)(3.83), Rheumatoid arthritis-Homo sapiens (human)(3.83), Focal adhesion-Homo sapiens (human)(3.79), Lysosome-Homo sapiens (human)(3.68), Chagas disease (American trypanosomiasis)-Homo sapiens expression.Then the result could be used to study the mechanism of the differential genes.Method:1.The method of how to screening the differential genes needed to be verified by RT-PCR: We compared the results of the gene expression microarrays and the 52 differential proteins screened by our research group through the experiment of proteomics. The genes, which have similar variation and the greater difference relatively were screened. At last 15 genes were found to verified.6 genes up regulated among them are:GNB2L1、ARHGDIB、 STMN1、LGALS7、 EIF5A、 TAGLN. On the other hands,9 genes down regulated among them are:S100A7、 S100A8、S100A9、 ANXA1、 ANXA2、ANXA5、CSTB、CRYAB、SERPINB3.2.32 paired tissues of the oral squamous cell carcinoma and the tissue by the side of carcinoma removed in 2013,were collected to do the RT-PCR expression verification experiment, which are a little greater sample numbers relatively.Result:15 differential genes,which have similar variations and the greater difference relatively in 32 paired tissues of the oral squamous cell carcinoma and the tissue by the side of carcinoma were screened to do the RT-PCR expression verification experiment. As a result, all the differential genes verified among 32 tissues have statistical differences(P<0.001). That confirmed the reliability of the gene expression microarrays again.Conclusion: Fluorescent semiquantitative RT-PCR technique is the mainly verification of the genes microarrays. In this experiment, we verified the differential genes expression among 32 tissues through fluorescent semiquantitative RT-PCR technique. The results show the consistent of the gene expression microarrys and the fluorescent semiquantitative RT-PCR. The Roche NimbleGen all gene expression microarrays have high reliabilities according to the results of consistent of the gene expression microarrys and the fluorescent semiquantitative RT-PCRChapter 4 The construction of relationship among the differential genesPurpose:15 differential genes, which have similar variation and the greater difference relatively, screened by comparing the results of the gene expression microarrays and the 52 differential proteins screened by our research group through the experiment of proteomics in advance, were input to the database of STRING to construct the net work of the relationship by the way of the Bioinformatics. And analysis the function,3-dimension structures, structure regions and the phosphorylation position of the node genes through NCBI, NEXTPROT, EMBL-EBI, NetPhos2.0 databases. And the result maybe provide some clues for the experiments followed-up.Method:l.The confirmation of the differential genes:19 differential genes, which have similar variation and the greater difference relatively, screened by comparing the results of the gene expression microarrays and the 52 differential proteins screened by our research group through the experiment of proteomics in advance.At last 19 genes were found to verified.6 genes up regulated among them are:GNB2L1、 ARHGDIB、STMN1、LGALS7、EIF5A、TAGLN. On the other hands,9 genes down regulated among them are:S100A7、S100A8、S100A9、ANXA1、ANXA2、ANXA5、 CSTB、CRYAB、SERPINB3.2. The differential genes were inputted to the database of STRING (http://string.embl.de) to analyse and construct the net work of the relationship by the way of the Bioinformatics and to find the possibly relationship among the protein subunits.3. Analysis of the node genesResult:1. Construct the net work of the relationships among the 15 differential genes by the STRING database. Among the complicated interactions network of 15 differential genes, the TAGLN、LGALS7、SERPINB3 and GNB2L1 have the most interactions which called the node genes.2. The node gene of TAGLNTAGLN gene interact with 9 differential genes through 18 interactions:COG4826(SERPINB3)、COG2319 (GNB2L1)、KOG1997 (STMN1) KOG2392 (SERPINB3)、KOG3587 (LGALS7)、KOG2046 (TAGLN)、KOG3205 (ARHGDIB)、KOG3591 (CRYAB)、KOG0819 (ANXA1、2、5)、 NOG26977 (ANXA2)、NOG26977 (ANXA2)、COG5199 (TAGLN)、KOG1997 (STMN1)、 KOG2392(SERPINB3) KOG3587(LGALS7)、KOG3591 (CRYAB).KOG0819 (ANXA1、2、5)、KOG3205 (ARHGDIB)3. The node gene of LGALS7LGALS7 gene interact with 9 differential genes through 10 interactions:KOG0819 (ANXA1、2、5)、KOG2046 (TAGLN)、KOG3591 (CRYAB)、 KOG3205 (ARHGDIB)、KOG2392 (SERPINB3)、KOG1997 (STMN1)、 KOG0279 (GNB2L1)、NOG26977 (ANXA2)、NOG29074 (CSTB)、COG5199 (TAGLN).4. The node gene of SERPINB3SERPINB3 gene interact with 9 differential genes through 10 interactions:KOG0279 (GNB2L1)、KOG3205 (ARHGDIB)、KOG2046 (TAGLN)、 KOG0819 (ANXA1、2、5)、 KOG3591 (CRYAB)、KOG3587(LGALS7)、 NOG29074(CSTB)、NOG26977(ANXA2)、NOG47012(S100A9)、 COG5199(TAGLN).5. The node gene of GNB2L1GNB2L1 gene interact with 7 differential genes through 9 interactions:COG5199 (TAGLN)、COG0231 (EIF5A)、 COG4826 (SERPINB3)、 NOG26977 (ANXA2)、 NOG75290 (STMN1)、 NOG29074 (CSTB)、 KOG3587 (LGALS7)、KOG2392 (SERPINB3)、 KOG3271 (EIF5A).Conclusion: We construct the net work of the relationships among the 15 differential genes by the STRING database. Among the complicated interactions network of 15 differential genes,the TAGLN、 LGALS7、 SERPINB3 and GNB2L1 have the most interactions which called the node genes. So we thought the 4 differential genes are the key nodes of the OSCC, which agreement with the other researchers. Following which the farther confirmatory experiments would be done to make sure the mechanisms of these differential genes according the results of the research.Research innovation1.We use the Gene Expression Microarrays to get the expression and variety of the whole genome of the oral squamous carcinoma tissue,and check the variety of gene related to the arisen and development of the oral squamous carcinoma. The results could be used to investigate the mechanism of oral squamous carcinoma.Then the methods of the molecular biological and information biology are used to validate the differential genes of oral squamous carcinoma by large samples relatively.In that case,the conclusion will have more persuasion.2.Refer to the data results of the gene expression microarrays and the results of the differential proteins of oral squamous carcinoma by our research group experiments using the methods of proteomics in advance.As a result,we chose 15 differential genes validated by the RT-PCR to find the differential genes and proteins,and the relationship among them. And analysis the function,3-dimension structures,structure regions and the phosphorylation position of the node genes through NCBI, NEXTPROT, EMBL-EBI, NetPhos2.0 databases, And the result maybe provide some clues for the experiments followed-up.3.Our experiment integrate the new technical of gene microarrays and proteomics to study the mechanism of oral squamous carcinoma.and "research in group"is the characteristic."research in group"means the study don’t limit to single gene or protein.It focus on the whole relationship network related to the genes or protein. |
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