The Cloning And Functional Research Of Differential Genes Between High Malignant Bladder Transitional Cell Carcinoma And Its Normal Epithelium

The occurrence and development of bladder transitional cell carcinoma (BTCC) is related with the mutation and abnormal expression of many genes. It is necessary that clone and identify BTCC-specially expressed genes to elucidate the mechanism of BTCC. Attempts have been made to isolate genes over expressed in cancer cells with methods such as representational difference analysis (RDA), subtractive hybridization, differential display of mRNA (DD), and differential subtraction display (DSD). Although these methods have been proved to be powerful tools for the construction of gene indexes, they are not excellent enough on account of their low specificity and sensitivity.Suppression subtractive hybridization technique was used to construct a cDNA subtractive library of human BTCC, Dot blot was then used to screen the genes that differently expressed between BTCC and it's normal epithelia tissues. Some clones in the library were selectedrandomly and sequenced. Then the results were analyzed with the Blastsoftware. The novel gene which had many copies in the library was identified its expression in BTCC and normal epithelia tissues by Northern blot. For the novel gene fragment that was specially expressed in BTCC, we used SMART RACE technology to clone its full length and also used fluorescence immunology situ hybridization to identify its chromosome location. After transfecting the antisense oligodeoxynucleotide of BQ135232 into bladder carcinoma EJ cell line, the proliferation activity, growth speed, apoptosis, and mortality changes in EJ cell were determined. Finally, the expression of BQ135232 in different stages and grades of BTCC tissues were studied by the assay of RT-PCR.The results showed that human BTCC subtractive library with highly subtractive efficiency was set up successfully. The amplified library contains 376 positive clones and 83 negative clones. Random analysis of 100 clones with restriction enzyme digestion shows that 89 ones contain inserts. After screened by Dot blot, 76 inserts of all the 89 PCR-amplified ones were real positive. Sequencing and analyzing with BLAST, all these 76 inserts represented 23 kinds of known genes and 5 anonymous ESTS in which the fragment of BQ135232 has 3 copies. Northern blot analysis showed that BQ135232 expressed higher in bladder carcinoma tissues or cell lines than other tumors or cell lines. Byusing Smart Race technique, we obtained the full length of the novelgene that was about 1.5kb long. The expression of BQ135232 was higher in higher grades and stages of BTCC than in lower ones. After transfection of BQ135232 antisense oligonucleotide the mortality of EJ cell increased evidently, the proliferation activity and growth speed were inhibited remarkably at the same time. Also the antisense oligonucleotide can induce the apoptosis of EJ cell.Our research indicated that BQ135232 is an important novel gene related with the generation and development of BTCC. Its over expression could stimulate the growth and proliferation activity of cancer cells and played an anti-apoptosis effect in BTCC. Transfection of antisense oligonucleotide could inhibit the development of BTCC. The study of this gene provided a new clue for research of BTCC, and also provided an instruction for the diagnosis and therapy of BTCC. The construction of highly efficient cDNA subtractive library of human BTCC lays solid foundation for large scale screening and cloning of new and specific oncogenes or tumor suppressor genes of BTCC. The novel specially expressed genes provided an important clue for researching the mechanism of the occurrence and development of BTCC.