Biological Properties Based On Bioinformatics Analysis Of EML4-ALK Fusion Gene In Non Small Cell Lung Cancer And The Functional Validation For STAT3of ALK Downstream

BackgroundLung cancer is the leading course of mortality in oncology with heterogeneity, and majority of the cases are non-small cell lung cancer (NSCLC). The selections of treatments are based on the histological classification, which has been used for decades. Such classification has meet ceiling effect now. Base on the advantage technology of molecular biology, the classification of lung cancer in molecular levels is extremely urgent. To choose treatments depending on the EGFR mutation status has received extensive survival benefit for the patients who are suitable for such therapy. Such strategies have been the paragons for lung cancer treatments. However, there are still many lung cancer patients that we do not know if there would be suitable targets for them, so we need to investigate more exactly classification in molecular level for NSCLC.The anaplastic lymphoma kinase (ALK) gene encodes a receptor tyrosine kinase (RTK) that has been discovered to be present in a number of fusion proteins consisting of the intracellular kinase domain of ALK and the aminoterminal portions of different genes. The ALK-EML4fusion attaches the ALK gene to a gene involved in microtubule formation and stabilization,"echinoderm microtubule associated protein-like4"(EML4). This fusion generates a transforming fusion tyrosine kinase. several isoforms of which have been identified in lung cancers. Published studies using reverse transcriptase-polymerase chain reaction (RT-PCR) analysis or fluorescent in situ hybridization (FISH) to characterize EML4-ALK fusions in lung cancer have revealed frequencies ranging from0.5%to7.5%in general lung cancer patients. Importantly, in some cell lines harboring EML4-ALK fusions, targeting of ALK using specific inhibitors has shown promising efficacy for treatment of lung cancer, so as in some early clinical trials.EML4-ALK defined a new kind of NSCLC and the fusion gene plays important roles in carcinogenesis of lung cancer. Specific inhibition of ALK would receive encourage clinical benefit for patients who harbor the EML4-ALK fusion gene. From the above. EML4-ALK has the promise as a new therapeutic target. Based on the research conduncted by our institute, we found that in our country the distribution of ALK fusion gene in NSCLC was more frequent.12samples in103continued samples were found to harbor EML4-ALK fusion gene with different variants, which was present at a frequency of11.6%in total samples, including4variant Ⅰ;1variant Ⅱ;3variant Ⅲa;1variant V and3variant Ⅵ. And all kinds of EGFR mutations were34. which in total was present33%of all detected samples. All kinds of K-RAS mutations were6cases, which was present5.8%of all samples. Besides one EML4-ALK fusion gene; there were no other EGFR or K-RAS mutation were detected in another11case harboring EML4-ALK. Also we found In Chinese NSCLC patients, who are EGFR wild type, non-smoker or adenocarcinoma were higher incidence to harbor EML4-ALK. EGFR, K-RAS and EML4-ALK were mutually exclusive. Base on the molecular epidemiologic investigation of EML4-ALK, we supposed ALK TKIs maybe a gift special for Chinese lung cancer patients. But the mechanism how ALK transformed normal cell into cancer cell and the relationship between EML4-ALK fusion gene and other molecular downstream are still unclear;the mechanisms of signal transduction in ALK downstream are also unknown. Base on the data from epidemiology, we need to conduct deeply research concerning two areas below:Part one:comprehensive analysis of the changes of EML4-ALK fusion gene singal pathway would be conducted through bioinformatics methods. The differentially expressed genes would be screened out. annotated and categorized into Gene Ontology. In this part, we found STAT3was a key gene in the development for lung cancer harboring EML4-ALKPart two:functional validation for STAT3. STAT3was hypothesized to play a key roll in many biology processes and molecular functions. For validation, RNAi was conducted on model cell lines harboring EML4-ALK or not. Also we contribute an overexpression cell model for STAT3in cell line H2228, Comprehensive cell functional changes were analysis.Research design and Primary ConclusionPart one. Research on Molecular Mechanism of NSCLC harboring EML4-ALK Based on BioinformaticsChapter1. The relationship between ALK expression level and EML4-ALKObjectiveTo find out a suitable algorithm that could extract microarray signal data from CEL. The relationship between ALK or EML4and EML4-ALK was also be analyzed.MethodsAfter finishing the microarray. Robust multiarray analysis algorithms were chosen to extract the signal data from RAW files. One-way ANOVA was used to analyze the difference among EML4-ALK fusion group, non-fusion group and normal tissues; while Pearson chi suquare test was used to analyze the correlationsResults and ConclusionBesides the reference gene designed by Affymetrix,18123genes were annotated via customCDF. The average level of ALK expression in EML4-ALK fusion group was5.44, while the non-EML4-ALK fusion group was2.99and2.86in normal tissues. And the average level of EML4expression in fusion group was6.47, while the non-fusion group was6.87and6.42in normal group. The fusion of EML4and ALK were significant correlation with expression level of ALK.Chapter2. Screening of the differential expression genes and functional annotationObjectiveTo find out the differential genes between EML4-ALK and wild type NSCLC through SAM method. Gene ontology was referred to annotate the functions of genes. And then the differential expression genes were enriched base on the biology process, molecular function for the functional validation; also the differential genes were enriched into pathway base on the data base from KEGG and BioCarta.MethodsBRB-Array tools were used to find out the differential genes between EML4-ALK and wild type NSCLC. Database for Annotation. Visualization, and Integrated Discovery (DAVID) system were used to annotate the genes function into different categories. Data base based on gene ontology, KEGG, BioCarta were used to enrich the differential genes into GO annotation and pathwaysResultsBetween EML4-ALK and wild type NSCLC, there are83differential expression genes; compared with wild type, including53genes down regulation and30genes up regulation. These genes were annotated into50different GO biology process and15pathways.ConclusionAll the differential expression genes revealed a correlation, most of them composed into group to play biological functions. There are group of molecular changed during the development of EML4-ALK, the gene changed in EML4-ALK NSCLC effected with each other.Part two. Functional Validation of STAT3Chapter1. Contribution of the protein-protein interaction of STAT3in NSCLC ObjectiveTo contribute the network that represents the protein affected with STAT3in NSCLC. And investigate the key roll of STAT3in NSCLC with EML4-ALK fusion gene via protein level.MethodsRefer to the differential expression genes between wild type and EML4-ALK NSCLC, cytoscape was used to contribute the protein-protein interactions base on the data base of SWISS-PORT, HPRD and literature mining. The differential molecular function and pathway were compared between EML4-ALK and wild type NSCLCResultsThere are113proteins which interact with STAT3in NSCLC. involving at least24pathways highly correlated with tumorigenesis and development. All of113genes were functional clustered and compared with the gene sets which are exclusive in EML4-ALK NSCLC patients. The proteins interaction with STAT3which also appeared up/down regulated in EML4-ALK NSCLC were involved in the cell cycle, transcription regulation and tumor proliferation.ConclusionFrom the protein interaction network, we found the biology processes and molecular functions STAT3involved were cell cycle, transcription regulation and tumor proliferation, which showed abnormal in EML4-ALK NSCLC. Moreover. STAT3plays key roll in the pathway of Wnt and Notch, which are also found dysregulation in EML4-ALK NSCLC. The molecular functions of STAT3in EML4-AKL NSCLC need further validationChapter2. Effects of different expression level of STAT3in NSCLC model cellsObjectiveTo investigate whether our RNA interference method based on shRNA vetctor could induce gene silencing in different model cells, and to evaluate which oligo nucleotides could supply the best silencing effect; to observe the effect on cell biological characteristics when STAT3was knocked down. The relationship between signal transduction involving STAT3and the cell proliferation, cell cycle alteration. apoptosis after STAT3RNA interference were analyzed. To investigate whether our overexpression method based on transient transfection vetctor could induce overexpression of STAT3in model cell, and to evaluate when increasing STAT3gene expression. the effect on cell biological characteristics. The relationship between signal transduction involving STAT3and the changes of cell proliferation, cell cycle and apoptosis after STAT3overexpression were analyzed.MethodsRecombination and identification for pSilencer4.1CMV-shRNA-STAT3:firstly, synthetizing the template oligonucleotides getting from RNAi Consortium; then ligating the annealed shRNA-STAT3oligo and inserting into the vectors; thirdly, transforming E. coli with the ligation products and purifying recombined plasmids; fourthly, identifying the positive clones through direct sequencing:fifthly. proliferating and storing the correct plasmids for transfection.Transient transfection:the recombinat pSilencer4.1vectors and the control vectors were transfected into model cell lines with Fugene6.Effect of STAT3knocked down:Western Blotting was conducted to evaluate the reduction degree for the expression of the STAT3protein.Investigation of the changes for biological characteristics after STAT3was knocked down:Flow cytometry and Western Blotting were used to evaluate cell status for apoptosis after RNA interference.Construction and identification of pcDNA3.1+STATS:A series of procudures (including construction of expression vector, validation and amplification of plasmid including STAT3, transform E. coli with the gene, purify plasmid for transfection and identify clones with the STAT3template insert through direct suquencing) were performed to construct and identify the overexpression vector.Transient transfection:pcDNA3.1+STAT3and the control vectors were transfected into model cell lines with Fugene6. Effect of STAT3overexpression:Real Time qPCR and Western Blotting was used to measure the increasing in the expression level for STAT3.Investigation for the changes of biological characteristics after the expression level of STAT3was up-regulated:Western Blotting was used to evaluate the relative marker concerning cell proliferation, apoptosis and cell cycle distribution after STAT3overexpression.ResultsEffect of STAT3knocked down:In H2228and H1299cell line, the STAT3protein expression was down regulated for64±2.3%and52±3.7%by shRNA-STAT3-1and shRNA-STAT3-2respectively through Western Blotting.For signaling analysis, the decresing of STAT3related to a down regulation of mTOR expression in protein level.Alteration of biological features after STAT3was knocked down in H2228cells: Apoptosis analysis by flow cytometry showed that shRNA-STAT3induced early apoptosis, while there are no significant changes in cell line H1299Effect of STAT3overexpression:In H2228cell line, the STAT3transcription expression was up-regulated significantly and the protein expression was increasing, respectively through Real Time qPCR and Western Blotting.For signaling analysis, the increasing of STAT3related to a slightly up-regulation of mTOR expression in protein level and the significantly increasing of phosphorylation of mTOR could be observed.ConclusionThe specific shRNA-STAT3we constructed showed a significant interference effect for down-regulation of STAT3expression, inhibiting the proliferation and inducing apoptosis in H2228cells. To knockdown the expression of STAT3in ALK fusion positive cells might provide a new method for gene therapy and research of NSCLC The transient transfection for overexpression vector of STAT3showed a remarkable effect in up-regulation of STAT3expression, which might increase the cellular proliferation and motilit in H2228cells through the enhancement in mTOR pathway.