High Throughput Screening Of The Susceptible Genes Of Human Bladder Cancer And The Case-Control Study Of 45 Patients

BackgroundThe incidence of bladder Cancer is the 9^th of all the malignant tumors all around the world.It is the 8th frequent Cancer in Chinese males while behind the 12th in Chinese females.With the industrialization and food and drink habit changes in China in recent years,it has been reported that the incidence of bladder cancer in several Chinese cities have the tendency of increasing.The carcinogenesis of bladder cancer is a very complicated pathological process which involves several factors and steps.Some of the factors are genetics,some are environmental factors.Epidemiologic study showed that smoking and long-term exposure to industrial chemistry products are the two major risk factors for the pathopoiesis of bladder cancer.The canceration of normal bladder cells started from the DNA and gene expression level changes.While the traditional single factor analystic model and the single level analystic model of genome,transcriptome or protein translation level couldn't systemically elucidate the characteristic and regularity of the pathogenetic of bladder cancer,it also couldn't make a scientific evaluation and analysis of the final turnover of early pathological changes of the mucous membrane of the urinary bladder.With the development and build up of the bio-chip technology which could be applied for large scale and high throughput analysis of genes and proteins,as while as the consummate of the bioinformatics methods,it became possible now to study the pathogenesy of bladder from multiple factors and multistrata.Meanwhile,the invention of real-time PCR technology had improved the PCR technology from qualitation to quantitation.It also provided a higher specificity,lower contaminat and higher automaticity method than the common PCR,which has been applied in the quantition of PCR product of DNA and RNA,gene expression research, diagnosis of genetic disorders,detection of pathogens,etc.Real-time PCR has also been well applied for the quantitative verification of microarry hybridization data.Case-Control Study of epidemiology could be applied in studying of the genes as a possible biological exposure risk factor of the body itself,whether it has significant difference between the bladder cancer tissue and the normal mucous membrane of urinary bladder.How much is the correlate strength between the gene expression difference with the bladder cancer,how big is the percentage of attributable risk,etc.All of these are very important evidence for verifying the reliability of microarray data,as while as for screening out the most valuable genes for the hypersensitive target gene of bladder cancer.The successful screening of the gene expression profile and the tumor marker gene of bladder,together with the molecular mechnims research and epidemiological research of the bladder cancer and related diseases,and the early diagnosis by the introscopic method are the very promising way for overcoming the problems currently and to obviously improve the early diagnosis of bladder cancer. These could also win some significant result in the screening,diagnosis and therapy of bladder cancer,and later on have some virtual influence in the clinical treatment and prognosis of patients with bladder cancer.ObjectiveTo screen the gene expression profile of the Chinese bladder cancer patients by the genome wide DNA microarray,study the molecular mechanism of bladder cancer,screen out the tumor specific differentially expressed genes.Then verify the microarray detection data by applying the real-time PCR method in quantifying groups of large sample of bladder cancers and controls.Finally screening out the hypersensitive tumor-marker genes by the epidemiological Case-Control Study,which could be applied in the susceptibility prediction,early diagnosis,disease surveillance and molecular targeted therapy of bladder cancer.Methods1.Collection of clinical samples 10 cases of invasive bladder cancer and 10 cases of normal urinary bladder transitional epithelium for DNA microarray screening,45 cases of invasive bladder cancer and 45 cases of normal urinary bladder transitional epithelium for real-time PCR verification were aquired from NanFang Hospital of Southern Medical University.2.The extraction and quality arbitration of total RNAs from samples Total RNAs from samples were extracted by the Qiagen RNeasy mini kit and then quality arbitration was done by the lab on chip microarray with the Agilent 2100 bioanalyzer.3.Liner fluorescent amplification labeling and quality arbitration of RNA samples Total RNAs were labeled by the Agilent low input Liner fluorescent amplification kit and then quality arbitration of the labeled cRNAs were done by the lab on chip microarray with the Agilent 2100 bioanalyzer.4.Preparation,hybridization and screening analysis of the 70mer oligo microarray 70mer oligo probes from Qiagen were printed into microarrays by the Cartesian Pixsys 5500 microarray printer.The labeled cRNA samples were devided into 10 groups and applied to 10 microarrays for hybridization separately.Axon Genepix 4000A microarray scanner was applied for scanning and Genepix pro 6.0 software were used for analysis of the microarray hybridization results.5.Cluster analysis of the microarray hybridization data Cluster software were applied for the systematic cluster analysis of the microarray hybridization data, treeview software were applied to display the expression difference between the bladder cancer and normal urinary bladder transitional epithelium.6.Panther Pathway analysis of the differential expressed genes Panther Pathway analysis were applied for check the signal pathways which have been involved by the co-upregulated and co-downregulated genes of 10 groups microarray data.7.Real-time RT-PCR verification of the differential expressed genes Qiagen Rotor-Gene SYBR Green RT-PCR kit were used to set up the reaction of real-time PCR detection,and Rotor Gene 6000 real-time PCR amplifier were applied for verifying 6 co-upregulated genes.8.Epidemiology study of the gene susceptibility of bladder cancer8.1 Significance test of Case-Control Study(X² test) was applied to compare whether the exposure of the 6 co-upregulated genes have significant difference between the case group and the control group,whether there is a stantistical correlation between the high expression of the genes and the bladder cancer had also been judged.8.2 Odds ratio(OR) analysis was applied for aquiring the exposure ratio between the Case-Control group,so that it could show the strength of correlation between he high expression of the genes and the bladder cancer.95%confidence interval of OR was also appraised by the Miettinen algorithm.Results1.70mer genome wide oligo microarray were applied for screening the gene expression profile of 19568 genes in 10 pairs of bladder cancer and normal urinary bladder transitional epithelium tissue.7839 genes were identified as valid expressed in all these samples.152 genes were screened out as co-expressed differential genes, in which 70 genes were co-upregulated and 80 genes were co-downregulated.There are 23 genes in the co-upregulated genes and 17 genes in the co-downregulated genes were confirmed to have some correlation with tumorigenesis.2.Panther Pathway analysis showed that the 70 co-upregulated genes were involved in 37 known signal pathways while the 82 co-downregulated genes were involved in 32 known signal pathways,including some pathway which had direct correlation with bladder cancer or tumorigenesis. 3.Of all the 6 genes selected out for verification,X² value of FASN,STMN1,TYMS,VEGF,KRT19 were 51.61,51.61.71.43,61.95,64.21,which were much more higher than X² 0.01(l)=6.63,so that we got a conclusion that the expression level of these genes have significant difference between the case group and the control group.This proved the microarray data were true.Gene TYMS and VEGF were identified as higher correlation with bladder cancer,the OR value of these genes were 451and 238.86.Conclusion1.70mer genome wide oligo microarray were applied in this research for screening the gene expressiong profile of 19568 genes in 10 pairs of bladder cancer and normal urinary bladder transitional epithelium tissue.We had aquired the molecular marker profile of bladder cancer in Cantonese people,screened out 152 co-expressed differential genes of tumor,which were involved in 32 known signal pathways, including some pathway which had direct correlation with bladder cancer or tumorigenesis.These also proved that invasive bladder cancer is a complicated disease which involves multiple genes,multiple steps and multiple pathway regulations.The gene expression profile and tumor marker genes which were screened out by microarray are very important in the molecular mechanism and early diagnostic and therapeutic research of bladder cancer in Cantonese people.2.6 co-upregulated genes,FASN,STMN1,SPFNT2,TYMS,VEGF,KRT19 were selected out for quantification analysis in 45 cases and 45 controls by real-time PCR, Case-Control Study of epidemiology were applied for data analysis which had verified the data of microarray hybridization.Gene TYMS and VEGF were finally selected as the hypersensitive marker genes which could be applied in the susceptibility prediction,early diagnosis,disease surveillance and molecular targeted therapy of bladder cancer.3.The major problem of this project is that the incidence of bladder cancer is relatively low in Cantonese people,so that it's not easy to collect a large case group for research.The other thing is because of the genome wide microarray screening is to expensive for screening too many samples,we need to collect and screen some more samples in the future,to make this data mach more convincing.