Investigation Of A Novel Gene Mag-2 And Its Correlation With Tumor Metastasis

By virtue of two techniques, suppression subtractive hybridization (SSH) and DNA microarray, 79 ESTs were found to be expressed at least 2 times higher in PLA801D, a cell line with high potential of metastasis, than in PLA801C, a cell line with low potential of metastasis. These ESTs were supposed to be positively associated with the tumor' s metastasis phenotype. No. 83 EST was chosen for further study. The full length standed for by No. 83 EST, NM₀24293, were found by e-extension and BLAST. We named this novel gene mag-2 (metastasis-associated gene-2, mag-2) . Primary study showed that mag-2 could enhance invasion and metastasis of lung cancer cell by some unknown mechanism, indicating that mag-2 was a gene that could play a role in tumor metastasis process.Firstly we analyzed mag-2 bioinformatically. It is located at chromosome 2q35, and has a full length of 5. 2Kbp composed of 9 exons and 8 introns. Its open reading frame is 1536bp long, encoding a theoretical protein of 512aa with a MW of 55163. 7 Da. It is an acidic protein with an isoelectric point of pH4. 30. The analysis of this protein' s presumable secondary structure showed that it consists mainly ofα-helixes in addition to which littleβ-folds and random coil is found. It has 15 myristoylation sites,17 casein kinase II sites and 2 PKC sites. The attempts to surmise this protein' s tertiary structure and its function were frustrated because there has been no any known protein that has homological sequence with mag-2. The electronical tissue expression profile analysis told us that mag-2 was expressed in many normal human tissues. The mag-2' s expression ranks the highest in the following 10 issues: bone, fat, skin, testis, nerve, prostate, ovary, bladder, brain, parathyroideum gland. Mag-2 was predicted to be located in endochylema (the validity is 92.6%) .The detailed locations are as follows: endocytoplasmic reticulum(56. 3 %), vesica(21. 0 %),Golgi's body(10.3 %) , mitochondria (12.4 %) .In order to confirm mag-2' s full length of cDNA, the actual MW of the protein that it encodes, the accurate location and, most importantly,its function, we prepared and identified a polyclonal antibody specific to mag-2. Based on the results of antigen anticipation of mag-2 produced by bio-softwares, a mag-2-specific peptide of 17 amino acids was synthesized and conjugated with key hole limpet hemocyanin (KLH) to immunize Japanese rabbit. The antibody has a high tite, and is specific to mag-2, and can serve as a useful tool for us to study the function of mag-2. For the first time we confirmed experimentally that there is only ONE protein encoded by mag-2, and its MW is about 55kDa, which was the very MW of mag-2' s theoretically coded protein.. We also confirmed the difference in the expression level of mag-2 between PLA801C and PLA801D.On the basis of the results above, our study proceeded. Results revealed by a series of experiments show that mag-2 has but ONE transcript of 3.2Kbp, and is expressed in many, if we cannot say the most, human tissues. In the following 10 tissues the mag-2' s expression ranks the highest: nigra, salivary gland, cardiac apex, fetal liver, fetal spleen, left ventricle, pancreatic gland, bladder, jejunum and amygdala. And mag-2 was found to be expressed in many tumor cell lines that are of different tissue sources such as HepG2, MCF-7, HL-60, Hela, PG and BE1 as well as in many tumor cell lines that are of the same tissue source (lung) such as A549, H1299, Anip, PLA801D and H322. The protein Mag-2 was found, like small discrete particulates, to disperse in cell endochylema of PLA801D cells. And the very phenomenon was found in HepG2 cells. We failed to detect the existence of Mag-2 protein in the supernatant of either PLA801 C or PLA801D, indicating that this protein could not be secreted out of cells.As the most important part of our research, the correlation between mag-2' s function and metastasis phenotype of tumor cells was studied. We constructed a recombinant siRNA vector that was designed to inhibit the expression of mag-2 RNA, and PLA801D, the cell line that possesses a high potential of metastasis, were transfected with this kind of vector. A serial of cell biological experiments studied the phenotype changes associated with tumor metastasis. We found that, since mag-2' s expression was suppressed, the proliferation rate of PLA801D slowed down significantly, and that the ability of PLA801D to form colonies and to adhere to basal lamina was obviously weakened. Accordingly, we found that the suppression, of mag-2' s expression could also trigger off remarkable declination of the abilities of PLA801D to migrate and invade.And what' s more, we found that the downregulation of expression of both PCNA and MMP-2 resulted from the suppression of mag-2' s expression,indicating that mag-2 may exert its strength on metastasis of tumor cells by regulating their abilities of motion and adhesion. All of these data indicated that mag-2 gene plays an important role in tumor metastasis.