The Study On The Function Of Colorectal Tumor Related ST13 Gene And Protein

Colorectal tumor is the third of the ten leading cancer types with the percentage of 9-15%. Because most colorectal carcinomas appear to arise from adenomas, studies of different stages of colorectal neoplasia may shed light on the genetic alterations involved in tumor progression. The model of colorectal tumorigenesis in which the steps required for the development of cancer often involved the mutational activation of oncogenes coupled with the loss of several genes that normally suppress tumorigenesis. The changes of mismatch repair genes and the factors of cell cycle checkpoint also include in the model. But all of the molecular changes can not explain the whole process of colorectal tumorigenesis.In 1993, we reported the identification of a novel gene ST13 from a substractive cDNA library of normal intestinal mucosa, the expression of which was down-regulated in colorectal cancer by Zhejiang university cancer institute in the way of exploring new factors responsible for the progress of colorectal cancer. The study on the gene structure, chromosome assignment, RNA expression and its function showed that ST13 genomic DNA had 32017 base pairs, comprised of 12exons. Correspondingly its cDNA had 3127 base pairs , the open reading frame(ORF) was 1170 base pairs and coded for a 369-amino acid protein. The gene was located on human chromosome 22ql3. Northern blot analysis of RNA and immunochemistry analysis of protein from multiple human tissues suggested that ST13 was ubiquitously expressed. The expression of ST13 was various in different types of tissue and cell lines. STB protein was present only in the cytoplasm. In vivo experiment suggested that ST13 might be related to the growth and metastasis of tumor.In 1995, the Hsc70-interacting protein Hip was found. Hip participated in theregulation of the eukaryotic 70 kDa heat shock cognate Hsc70. Hip stabilized the ATP state of Hsc70 that had a high affinity for substrate protein. A 48-kDa protein(P48) that transiently associated with progesterone receptor was isolated in 1996. Immunoprecipitation of P48 complexes revealed a common association of P48 with hsp70 and, to a lesser extent, with hsp90 and hsp60. P48 was a novel component of the cytoplasmic molecular chaperone machinery. When three protein were compared, we found they were the same one protein.Hip regions included a glycine-glycine-methionine-proline(GGMP) tandem repeat, the C-terminal region, and a tetratricopeptide repeat(TPR). ST13 regions containing motifs shared by other proteina suggest that ST13 may take part in apoptosis and play an important role.The aims of this study are: First to detect the mRNA expression level of ST13 gene in colorectal carcinoma and the normal intestinal mucosa of the same patient, then to study the relationship between the expression level and the clinical pathological characteristics. Second to comform if the ST13 take part in cell apoptosis and it's function. The methods and results are in below:Part one: The expression of ST13 gene in colorectal tumor tissues and cell lines by real-time PCR.Chose 50 patients with colorectal cancer and 5 kinds of cell lines. The mRNA quantitative expression level of ST13 gene in colorectal carcinoma and the normal intestinal mucosa were detected by real-time PCR. The result showed that the ST13 mRNA expressed in the normal mucosa adjacent to colotectal carcinoma was higher than that of in colorectal carcinoma The result had significant difference in statistics(P<0.05). But compared the Duke's stages, lymphanode metastasis , differentiation level, the result had no significant diffirence in statistics. In the detected 5 cell lines, the expression of ST13 in normal intestine cell line NIC was almost the same as L174T, but was higher than that of SW480 and RKO. The expression of ST13in SW620 was the lest in all.Part two: Study of the function of ST13 gene by antisense transfection.Constructed antisense vector pcDNA3.1-/ST13, then transfected the vector into K562 cells. Chose six clones by G418 selecting an...