The Functional Prediction Of Cancer Associated Gene HMAT1 And The Silencing Effect Of Its SiRNA

Cervical carcinoma is one of the commonest malignant tumors that have great influence on women's health, and the incidence and the rate of death caused by cervical carcinoma in developing countries are very high. In China, Hubei, Jiangxi, and Xingjiang. Provinces are areas that have high-incidence of cervical carcinoma,It was previously believed that the infection of the high-risk type human papillomavirus (HPV) such as HPV16 or HPV18 is that the major cause of cervical carcinoma, because this kind of infection could be detected in more than 90% of patients with this cancer. But current research indicates that HPV alone cannot cause the carcinoma, and the experiments carried out in vitro shown that some cellular oncogenes are also needed to tranform the cells in addition to HPV infection. Based on these results, our group analyzed the cervical carcinoma specimen by using the oncogene and suppressor gene cDNA microarrays. Through these experiments, we find that the Homo sapiens homolog of mouse MAT-1 gene (HMAT1) is a candidate tumor oncogene in cervical cancer.In this study, we firstly performed some bioinformatic analysis on the HMAT1 gene to predict its characteristics. Blasting the gene sequence of HMAT1 against Genbank showed that the Homo sapiens PEA15 protein (PEA15) gene has a high identity (99%) with HMAT1. HMAT1 codes for a polypeptide with 75 amino acid residues. Amino acid composition analysis indicated that Lys(10.66%), Leu(10.66%), Arg(9.33%) and Ser(9.33%) have the highest percentage. Secondary structure analysis predicted that the a-helix are almost concentrated in the C-terminal, while the 3 -sheet are almost located in the N-terminal. The prediction of hydrophobicity region in 4-20aa was consistant with the predicted transmembrane region locating in 10-15aa. Signal peptide analysis by using SignalP Version 3 showed that there were potential cleavage sites in 21-22aa. It was predicted that 33-35aa, 45-47aa and 62-64aa were potential protein kinase C phosphorylation sites, and 22-25aa and 62-65aa were potential Casein kinase II phosphorylation sites. It is estimated that these sites may be involved in the regulation of HMAT1 protein.Secondly, in order to study the function of HMATl gene by using RNAi technology, we established a platform for siRNA screen via the vector-based method. Taking the advantage of EGFP gene, we constructed a fusion expression vector for the expression of HMATl and then designed three different siRNA vectors targeting different regions of HMATl. These two kinds of vectors were co-transfected into B16F0 cells. Both of the intensity of EGFP and the RT-PCR were used to evaluate the RNAi's efficiency of the silencing HMATl. The results revealed that: for the first siRNA, when co-tranfecting cells by using the expression vector and siRNA vector in a ratio of 1:3 or 1:4, the intensity of EGFP was obviously weaker than the control, and the density of the cell were on the same level. For the second and third siRNAs, compared with the controls, there were no significant differences of the intensity of EGFP when they were transfected in serial concentrations. The RT-PCR results indicated that the amount of HMATl mRNAs in the cells tranfected with the first specific siRNA was significant lower (P<0.05) than the controls. For the second and third siRNAs, the amount of the mRNAs did not have significant difference with the controls.This study demonstrated that the siRNA could effectively silence the expression of HMATl. Because the siRNAs used in this study was not chemically synthesized, they had little toxic effects on the cells, which provided an effective tool for the further study of the function of the HMATl gene in cervical cancer. In conclusion, this study successfully established the siRNA expression vectors that target different regions of the HMATl gene, and the EGFP expression system that provided us a convenient and fast platform for the screen of the siRNAs. We also identified a siRNA which can effectively silence the expression of HMATl by using this system.