1. Molecular Events During Gastric Carcinogenesis 2. The Function And Molecular Mechanism Of SMC4 In Lung Adenocarcinoma

Part ?:Molecular events during gastric carcinogenesisChapter 1:Whole genome gene expression profiling of gastric precancerous lesions and gastric cancerBackground:Gastric carcinogenesis is a complex process,involving multiple stages,factors and genes.In the human model of gastric carcinogenesis,from normal gastric mocusa to cancer requires a series of sequential phases:Normal gastric mocusa?Non-atrophic gastritis ? Multifocal atrophic gastritis ? Intestinal metaplasia ? low-grade intraepithelial neoplasia?high-grade intraepithelial neoplasia?Cancer.It is of great clinical and scientific significance to study the molecular biology of gastric precancerous lesions and gastric cancer comprehensively and systematically by using whole genome gene expression microarray.Aim:To study the similarities and differences of gene expression profiles during stages of gastric carcinogenesis.To screen the molecules that may play key roles in the process of gastric carcinogenesis and potential biomarkers for early detection of gastric cancer.Methods and materials:In this study,132 fresh gastric tissue samples at different pathological stages,including 20 low-grade intraepithelial neoplasia(LGIN),23 high-grade intraepithelial neoplasia(HGIN),19 early gastric cancer(EGC),4 advanced gastric cancer(AGC)and matched adjacent tissues were examined with Agilent whole genome gene expression microarray.To verify the expression levels of molecules discovered in microarray,50 formalin-fixed and parrffin-embedded(FFPE)tissue samples from patients with gastric cancer were examined with immunochemistry(IHC).Each slice contains normal gastric tissues,precancerous lesions and gastric cancer tissues at the same time.Results:The microarray data analysis showed that gastric precancerous lesions(GPLs)were similar to gastric cancer at molecular level:Many hallmarks of tumor cells,including the upregulation of DNA replication,cell division,cell cycle related genes and the downregulation of negative regulation of growth,branched-chain amino acid catabolic process related genes had presented in precancerous stage;compared to paired adjacent tissues,precancerous lesions and gastric cancer had similar differential expression patterns,that was genes differentially expressed in gastric cancer tissues had already differentially expressed in precancerous lesions consistently.However,there were differences between GPLs and gastric cancer at gene expression levels,which mainly related to immune response and cell adhesion.On the one hand,we found that 562 genes may be involved in the malignant transformation of gastric cancer,among which a tightly linked 8-gene module including CD40LG,CTLA4,GZMB,ICAM1,ITGAX,SELL,TGFB1 and TNF was in the core of the protein protein interaction network.The 8 genes involved in leukocyte and mononuclear cell migration,negative regulation of immune response and extracellular matrix organization were related to the overall survival of gastric cancer patients.On the other hand,in order to find potential biomarkers for early detection of gastric cancer,we selected 4 genes that were continuously upregulated during gastric carcinogenesis and performed IHC.The results showed that the protein expression levels and positive rates of CCL3 and CD300A continued to increase during gastric carcinogenesis;compared with adjacent tissues,the protein expression levels and positive rates of OSM and CHI3L1 in precancerous lesions and gastric cancer tissues were significantly increased;however,there was no significant difference in the protein expression levels and positive rates of OSM and CHI3L1 between precancerous lesions and gastric cancer tissues.The receiver operating characteristic(ROC)curve analysis showed that these four molecules may have potential clinical diagnostic value in early detection of gastric cancer.Conclusions:GPLs were similar to gastric cancer at molecular level,which indicated that precancerous lesions could be the basis of gastric cancer.However,there were differences between GPLs and gastric cancer at gene expression levels,which were mainly related to immune response and cell adhesion.These changes may be closely associated with the malignant transformation of precancerous lesions to cancer.The 8-gene module may play key roles in gastric carcinogenesis.CCL3,CD300A,CHI3L1 and OSM are potential diagnostic biomarkers in early detection of gastric cancer.Based on the multistage model of gastric carcinogenesis,the study of gastric carcinogenesis process by using gastric samples at different pathological stages plays important roles in illustrating the molecular mechanisms,discovering key molecules during carcinogenesis and potential biomarkers in early detection of gastric cancer.Chapter 2:TCR repertoires of gastric precancerous lesions and gastric cancerBackground:Immunotherapy based on tumour-infiltrating lymphocytes,which mainly depends on the interaction between antigens and TCRs,has demonstrated potential clinical application for the treatment of solid tumours.However,many basic properties of the TCR repertoire require clarification,and the changes in the TCR repertoire during carcinogenesis,especially during the precancerous stages,remains unstudied.Aim:To characterize the tissue-infiltrating TCR repertoire during gastric carcinogenesis and illustrate the relationship between the local molecular phenotype and TCR repertoire of the microenvironment.Materials and methods:This study examined 41 gastric tissue samples at different pathological stages,including low-grade intraepithelial neoplasia,high-grade intraepithelial neoplasia,early gastric cancer and matched adjacent tissues.We performed multiple polymerase chain reaction(PCR)and deep sequencing of TCR ? complimentary determining region 3(CDR3)chains on llumina Hiseq 2000 platform.Moreover,to illustrate the relationship between the local molecular phenotype and TCR repertoire of the microenvironment,a whole-genome gene expression microarray analysis of the corresponding gastric precancerous lesions(GPLs)and EGC tissues was conducted.Results:Our results showed that TCR repertoires overlap between gastric lesions and matched adjacent tissues gradually decreased during gastric carcinogenesis,providing evidence for antigen-driven clonal expansion.Accumulating lesion antigen-associated T-cell clones became highly expanded,and the frequency they occupied increased with tumour progression.Moreover,the frequency similarity of the highly expanded common T-cell clones between gastric lesions and the adjacent tissues decreased dramatically during stages of tumourigenesis.Furthermore,an integrative analysis of the microarray data and TCR repertoire variation index using the network-based Clique Percolation Method identified an 11-gene module that can predict the overall survival of gastric cancer patients.Conclusions:In conclusion,our results revealed the multistage heterogeneity of tissue-infiltrating TCR repertoire during carcinogenesis.The interactions between mutated cells and the immune system may play pivotal roles throughout malignant transformation.Besides,the study improves our understanding of immune responses during gastric carcinogenesis.Part ?:The function and molecular mechanism of SMC4 in lung adenocarcinomaBackground:Mounting lines of evidence suggest that embryo developmental processes and tumorigenesis share many similarities in tissue organization,cellular biological behaviors and gene expression patterns.The re-examination of distinctive processes involved in normal development might help elucidate the intrinsic features of cancer that are significantly obscured by its heterogeneity and disorder.Structural maintenance of chromosome 4(SMC4)is a core subunit of condensin complexes that mainly contributes to chromosome condensation and segregation.In our previous study,the microarray data analysis of the human lung developmental tissues at four different stages and lung adenocarcinoma(ADC)tissue showed that the mRNA expression level of SMC4 was gradually decreased during lung development and increased in lung ADC.Aim:To explore the function and molecular mechanism of SMC4 in lung ADC.Methods and materials:We first performed real-time PCR assay of 43 paired lung ADC tissues and adjacent lung tissues to evaluate the mRNA expression level of SMC4.Further,a tissue microarray including 75 paired FFPE lung ADC tissues and adjacent lung tissues tissues was used to detect the protein expression level of SMC4 by IHC,the association between SMC4 expression and lung ADC prognosis were analyzed by the log-rank test.The effect of SMC4 on proliferation and invasion of A549 cells were measured using the Cell Counting Kit-8(CCK-8)assay and transwell invasion assay,respectively.To further investigate the molecular mechanism of SMC4 in cancer progression,we performed co-immunoprecipitation(Co-IP)coupled mass spectrum studies and identified DEAD-box helicase 46(DDX46)potentially interact with SMC4.To validate the results,reciprocal Co-IPs and double immunofluorescence staining in A549 cells were conducted.Results:SMC4 was upregulated in lung ADC tissues compared with matched adjacent tissues at both mRNA and protein expression level,validated by real-time PCR and IHC.High protein expression of SMC4 was an independent prognostic predictor of lung ADC(log-rank test,p=0.036).Cell experiments showed that SMC4 silencing inhibits the proliferation and invasion of A549 cells;besides,the expression of mitosis-related protein PLK1 and its downstream substrates cyclinBl and CDK1 was decreased.The microarray data analysis of lung development samples at four different stages and lung ADC samples showed that the mRNA expression pattern of DDX46 was similar to that of SMC4.Moreover,Spearman's correlation coefficient showed that SMC4 is positively correlated with DDX46 at the mRNA levels(r=0.629,p<0.0001).Reciprocal Co-IPs validated that SMC4 interacted with DDX46.Immunofluorescence co-localization experiments showed that SMC4 and DDX46 co-localized in each phase during the cell cycle.Conclusions:SMC4 was upregulated in lung ADC tissues compared with matched adjacent tissues.Besides,high protein expression of SMC4 was an independent prognostic predictor of lung ADC.SMC4 promoted the proliferation and invasion of lung adenocarcinoma cells.SMC4 interacted with DDX46,which may be involed in lung adenocarcinoma progression through affecting pre-mRNA splicing.Genes with similar expression pattern to SMC4 in development may also participate in tumorigenesis.The identification of genes that are essentially involved in development through a comparative study between development and cancer would be a practical strategy for discovering potential therapeutic targets and biomarkers and obtaining a better understanding of the mechanisms of carcinogenesis.