| Part ?:Correlation analysis between proliferation genes in lung development and prognosis in lung adenocarcinoma and lung squamous cell carcinomaBackground:The intimate connection between tumorigenesis and developmental processes has been studied for many years.There are clear analogies between cancer and development.Several studies have explored the relationship between cancer and development on the gene expression level and have suggested that there are some associations between gene expression during development and the clinical phenotypes of different cancers.It has important clinical and scientific value to find the relationship between tumorigenesis and developmental processes using gene expression profiling chip technology.Our group previously reported on the gene expression profile signatures of human lung tissues at 4 developmental stages(including whole embryos,early embryonic,middle embryonic lung,and mature lung),and we got 213 genes(named PTN1)which expression decreased steadily during lung development,but expression increased in lung adenocarcinoma(ADC).The increased expression of PTN1 genes were significantly correlated with the prognosis of lung ADC.Aim:To explore the association between the expression of development genes and prognosis in lung ADC and lung squamous cell carcinoma(SCC),and to reveal the biological mechanisms of the correlation.Methods and materials:69 paracancerous samples of lung SCC were examined with Agilent whole genome gene expression microarray.The altered gene map of lung development-lung cancer was constructed based on the existing data(including whole embryos,early embryonic,middle embryonic lung,and mature lung),the change pattern of 213 PTN1 genes were observed in this map.Downloading 4 independent sets of lung ADC microarray data,4 independent sets of lung SCC microarray data,and their corresponding clinical information were collected from GEO,these data were used to verify results.Prognosis was evaluated by the log-rank test.Correlations between genes were evaluated using the Pearson correlation test.Significant differences in the correlation coefficients between lung ADC and SCC were evaluated using a 2-way ANOVA.Graphical visualization of the genes interaction was generated using Cytoscape software.Results:Survival analysis showed that the expression levels of PTN1 genes were associated with survival in lung ADC patients but not lung SCC patients.All of the lung ADC datasets contained a set of highly correlated genes from PTN1 genes,but the lung SCC datasets had no similar set of genes.We identified 63 unique core genes from the PTN1 genes in the 5 lung ADC datasets,17 of these core genes appeared in at least 4 of the lung ADC datasets,and the 17 genes clearly interacted more strongly with each other in lung ADC than in lung SCC.Moreover,16 of the 17 core genes play major roles in the G2/M phase of the cell cycle.Conclusions:These data indicate that the expression of proliferation-related genes in lung development was significantly associated with survival in lung ADC patients,the synergistic effects of the 17 core genes play an important role in lung ADC prognosis.These genes may have significant clinical implications for the treatment and prognosis of lung ADC.Part II:Analysis of the expression profile of epithelial-stromal interaction in colorectal cancerBackground:Carcinoma is a complex system,including not only parenchymal cells but also stromal cells,the stroma constitutes the tumor microenvironment.Stroma cells are including endothelial cells,carcinoma-associated-fibroblasts,adipocytes,immune cells and so on.The tumor microenvironment of colorectal cancer(CRC)plays essential roles in supporting the occurrence,progression,metastasis and drug resistance in CRC.Microdissection technology can be used to isolate specific cell groups from clinical or animal tissue samples for molecular analysis,and have become one of the main methods for obtaining high purity tumor epithelium and stromal cells.The previous transcriptome studies about tumor epithelium and stromal mainly focused on the different expression of genes,and did not systematically analyze the changes of the epithelial-stroma interaction in the process of carcinogenesis.Aim:To explore the changes of epithelium-stroma interaction when normal colorectal mucosa develops into colorectal cancer.Methods and materials:50 CRC and paracancerous tissue were collected,and the RNA was extracted from fresh CRC and paracancerous tissue,20 pairs of paired tissue with RNA integrity greater than 7 were selected to make frozen sections,then the paired tissue were microdissected to obtain epithelial and stromal cells,then RNA was extracted.Paired RNA with integrity greater than 7(10 pairs)were constructed library and examined with Illumina Hiseq4000.Matrix eQTL package was used to construct epithelium-stroma co-expression network,iGraph was used to computed the network degree for each gene in each network,RedeR package was used to visualize the network,SANTA package and four gene sets were used to systematically test for network functional enrichment.In order to verify the expression level of key molecules discovered in co-expression network and differential expression analysis,143 formalin-fixed and paraffin-embedded tissue sample from patients with colorectal cancer at stage II were collected to made tissue microarray and examined with immunochemistry.The characteristic gene sets of 26 immune cells and GSVA were used to evaluate the immune characteristics in epithelium and stroma of CRC and paracancerous tissue.Results:The epithelium-stroma co-expression network was constructed for CRC and paracancerous tissue,respectively.579324 and 3852706 significant co-expression associations were identified in CRC and paracancerous tissue networks,respectively,and self-loop relationships emerged in the both co-expression network.The self-loop relationships of CRC co-expression network was significantly more than that of paracancerous co-expression network,and the most significant network edges in CRC represented epithelial-stromal self-loops.In the CRC and paracancerous tissue networks,the most highly connected genes contributed to the co-expression networks primarily through their stromal expression.The functional enrichment showed that the CRC and paracancerous tissue networks both have their significant function.In order to verify the expression level of key molecules discovered in co-expression network and differential expression analysis,we selected 3 genes(FXYD5,ITGA2,RPS19)that were only found in CRC co-expression network and increased expression compared with paracancerous tissue and performed immunochemistry.The results showed that the protein expression level of FXYD5,ITGA2 and RPS19 were increased expression compared with paracancerous tissue,FXYD5 and RPS19 were found in epithelium and stroma.Unsupervised clustering of GSVA scores found the immune characteristics were different between CRC and paracancerous stroma and epithelium.Treg cell characteristics was the most obvious in CRC stroma.Variety of immune characteristics are positive correlation between epithelium and stroma,and the immune correlation in epithelium and stroma of CRC was significantly higher than that in paracancerous tissue.Conclusions:The structure and function of epithelium-stroma co-expression network,the immune characteristics of epithelium and stroma,and the immune interaction between epithelium and stroma in CRC all have changed compared with paracancerous tissue.Self-loop relationship is a characteristic of CRC co-expression network,but its biological significance needs to be further study.FXYD5,ITGA2 and RPS19 are self-loop genes which expression increased in CRC epithelium-stroma co-expression network,the role in the progression of CRC need to be explored. |
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