Analysis Of The Profiles Of Gene Expression In Colon Cancer By Using Cancer Genome Anatomy Project And RNA Interference

Part I Detection of the profiles of gene expression in colon cancer by using Cancer Genome Anatomy ProjectObjective To investigate the differential gene expression profiles between colon cancer and normal tissues for further identifying gene markers that may be useful in the diagnosis and treatment of colon cancer.Methods We used the data of serial analysis of gene expression(SAGE) and related tools from Cancer Genome Anatomy Project(CGAP) to compare differential gene expression in colon cancer tissues with normal tissues. Differential genes were further identified by using RT-PCR in colon cancer cell lines (SW1116, Lovo, HCT8 and Hce8693)and 20 specimens of colon cancer.Results (l)Two SAGE libraries of colon cancer (Tu-98, Tu-102) and 2 libraries of normal colon tissues(NC-l, NC-2) were analyzed. These libraries contained a combined total of 195160 transcript tags including 97071 tags of colon cancer and 98089 tags of normal tissues respectively, comparison between libraries of colon cancer and normal tissues showed that 216 tags were significant differentially expressed greater than 2-fold. There were 33 transcripts that were elevated in colon cancers by 10-fold and 16 transcripts by 20-fold compared with normal tissues. On the contrary, there were 54 transcripts that were decreased in cancers by 10-fold and 25 transcripts by 20-fold.(2)17 differential genes in colon cancer were selected to be further confirmed by RT-PCR in colon cancer cell lines (SW1116, Lovo, HCT8 and Hce8693) and 20 specimens of colon cancer with a positive rate ranging from 35.3% to 76.5% in the cell lines and 88.2% in the specimens, respectively. The results confirmed by RT-PCR are consistently corresponded to SAGE date.(3)The differential transcriptions including TGFβ1, PSMC4 and HSPCA were identified as overexpressed genes in 12/20(60%), 10/20(50%), 7/20(35%) of specimens, respectively, (p<0.01) by using semi-quantitative RT-PCR.Conclusions The results suggested that SAGE data of CGAP maybe rapidly and reliably screen the differential gene expression profiles of colon cancer. Further study on these genes may provide the gene markers for the detection and treatment of colon caner.Part II Effect of RNA silencing of TGFpi on the growth of colon cancer cells by RNA interferenceObjective To identify the functions of differential genes detected by gene expression profiles. We further inhibited the gene expression of TGFpi which is one of the differential genes in the Part I of our study by RNA interference to observe the changes in cell cycle progression, growth and tumorigenesis.Methods small interference RNAs directed to TGFpi were self-designed and in vitro synthesized, and then transfected cells with lipid/siRNA complex, changes of cell cycle progression, cell growth and tumorigenesis were then observed and analyzed by flow cytometry and soft agar colony assay.Results (1) We designed and in vitro synthesized two small interference RNAs (siRNA) -TGFpl-1 and TGFpi-2 against TGFpi, and then transfected siRNAs into cytoplasm of HCT116, the inhibition of TGFpi transcription was confirmed by RT-PCR.(2) The cell strains had a 59%-82.5% increase in S phase and a decrease in G2 phase compared with control group after transfection of siRNA against TGFpi. The soft agar colony assay further demonstrated that siRNA against TGFpi led to a significant decrease in colony formation as compared with control group.(3) TGFpi-1 seemed to be slightly superior to TGFpi-2 according to the results of analysis of cell cycle and colony formation, although there was no significant differentiation of inhibitory effect between the two synthesized siRNAs against TGFpi by using semi-quantitative RT-PCR. However, both of them could effectively arrest the cell cycle in S phase and decrease the cell growth and tumorigenesisConclusions Our results demonstrated that inhibition of gene expression of TGFpi may lead to arrest the cell cycle in S phase and further decrease of cell growth and tumorigenesis. The function of differential genes detected by gene expression profile...