The Selection Of Fhit-related Genes And The Identification Of The Part Of Genes

Fragile histidine triad (Fhit) gene, the first cloned and identified common fragile site gene, spans FRA3B Fragile area and is located in 3p14.2. Fhit is confirmed to be a tumor suppressor by using cell biology, oncomolecularbiology, transgenic and gene knock-out technologies, it is involved in tumorigenesis and tumor development. The deletion and methylation of Fhit gene and the expression reduction of Fhit protein occurred in about 70% of human epithelial tumors, particularly in those tumors resulting from exposure to environmental carcinogens. Although Fhit as a tumor suppressor through Fhit-substrate complexes has been reported, specific Fhit signal pathways have not been defined. In order to study the mechanism of Fhit in DNA damage response and the signal pathways involved, the selection and identification of the genes involved in Fhit before and after ionizing radiation(IR) by using gene-chips.First, HeLa cells transfected the vector alone(HA) or plasmid containing wild type Fhit(3-18) were collected at 6h after 10Gy of exposure, and this two cell lines were also collected without IR treatment. Total four cell lines were analyzed by using the gene chips of Human-6 BeadChip (Illumina Corp.), each of chip contains >48,000 gene information from RefSeq, and differential expressed genes of D/C(3-18/HA), F/E(3-18 IR/HA IR),E/C(HA IR/HA) and F/D(3-18 IR/3-18) were obtained. When P <0.05, 648, 2820, 2816 and 2825 differential expressed genes of D/C, F/E, E/C and F/D were selected.Then differential genes were analyzed by online chip datum analysis software from CapitalBio biological company, in which showed the information about signal pathways and Pvalue. Up-regulated genes of CT45-5, IL13RA2, MAGEA1, SPANXB1, MDC1 and SEMA3C and down-regulated genes of SPINK4, SPINK6, IGFBP3, DUSP6 and IL8 were identified in D/C(3-18/HA), otherwise up-regulated genes of CDC2, YWHAG, CCNB1 and KPNA2 were identified in F/D(3-18 IR/3-18).The differential expressions of mRNA in these cell lines were verified, the results showed that the RT-PCR results were consistent with the results of the chips, except IGFBP3. Then the differential expressions of the protein in these cell lines was verified, because the commercial antibodies against the part of these proteins could not been ordered, we prepared polyclonal antibodies against CT45-5, MDC1 and SPANXB1. The titer and specificity were tested by ELISA and Western blot, the results showed that the titer of these antibodies were about 1:12800, and CT45-5, SPANXB1 and MDC1 expressed in prokaryotic cells can be recognized specifically by this antiserum, the expressions of CT45-5 and MDC1 in the cells were identified, the results were consistent with the results of the chips.In this study, we screened the genes which may been affected by Fhit gene by gene-chip technology, and a number of genes were screened, it is to provide a large amount of information and it is the basis to understand the role of Fhit in tumorigenesis and tumor development.