Investigation Of The Relationship And Molecular Mechamism About Mag-1 To Tumor Metastasis

Tumor metastasis always is a trouble problem in tumor prevention and therapy. Only when metastasis successfully blocked, the complete cure of malignant tumor can be reached. In the course of tumor development some tumor cells acquire invasive and metastatic potency which were induced by genetic alteration of tumor cells. It is a hot spot of research to search for the metastasis associated genes and reveal the molecular mechanisms of metastasis.Suppression subtractive hybridization (SSH) combined with DNA microarray is an effective method to isolate differential phenotype associated genes. With SSH method we isolated 79 differentially expressed sequences associated with highly metastatic phenotype and found sequence No.3 is completely homologous to the 3'end of NM₀32717 gene. NM₀32717 gene was identified by a Japanese research group who analyzed the differential expression profiling between hepatoblastomas and normal liver cells. However, the structure and function of the gene product is not exactly known. Hence we will investigate the relationship and molecular mechanism about mag-1 to metastasis in this thesis.We first measured the migrant behavior and the expression level of Id-1, a marker of metastasis, of PLA801C and PLA801D cells. The results show not only the migration but also the Id expression level of PLA801D are higher than that of PLA801C which confirmed that the cells used as tester and driver in SSH are a pair of highly and poorly metastatic tumor cells respectively. We also examined the transcriptive and translational level of mag-1 in PLA801C and PLA801D by RT-PCR, Western-blot and immunocytochemical staining. The results coincidence with the observation through SSH that mag-1 expression level is higher in highly metastatic PLA801D than in poorly metastatic PLA801C. In addition, we detected the higher expression level of mag-1 in paired highly than poorly metastatic tumor cells for example MDA-MB-231 and MCF-7 as well as BE and PG. Thus mag-1 may be a metastasis associated gene. We tested the effect of mag-1 through DNA transient transfection and found the expression of mag-1 in cells transfected with mag-1 sense vector increased but had no obvious change in cells transfected with mag-1 antisense vector. Therefore, the poorly metastatic tumor cells PLA801C and MCF-7 transfected with mag-1 sense vector and the stable population of cells that express exogenous mag-1 were selected in this paper. With the stably transfected cell lines,we tested the effect of mag-1 on tumor cells behaviors and found the morphology and proliferation of cells overexpressing mag-1 had no alteration but the migration, adhesion and invasion of cells transfected with mag-1 sense vector enhanced as compared with cells transfected with control vector.RNAi was utilized extensively to interfere with target gene expression. Since antisense vector can't inhibit the target gene expression efficiently, we employed RNAi technique to investigate the association between mag-1 and metastasis. We first constructed siRNA expression vectors that aim at mag-1, and then transfected PLA801D with the mag-1 RNAi vector. The cells in which mag-1 was inhibited showed a lower level of the migraant, adhesive and invasive behaviors. These results suggest that mag-1 is associated with metastasis and can facilitate tumor metastasis.In order to investigate the underlying mechanism through which mag-1 improve tumor metastasis, we observed mag-1 overexpression could not alter the expression level of PCNA, MMP-2, nm23H1, VEGF, MTA-1, Id-1, p53 and the concentration of Ca2+. While investigating the function of mag-1, we also pay close attention to the scientific advance in view of mag-1. Professor Yu Long , School of Life Science of Fudan University, cloned lysophosphatidic acid acyltransferase (LPAAT)-theta that belongs to a new member of LPAAT family in 2006. Mag-1 was found to share 100% identity with LPAAT-theta by blast which help us recognize the biochemical nature of mag-1. LPAATs catalyze LPA to PA and PA is a kind of important signal molecule. Recent observation indicated that PA can activate mTOR signal pathway directly. In our experiment we found that mag-1 overexpression did induce phosphorylation of S6K1 and 4EBP-1 which results from mTOR activation and the phosphorylation of S6K1 and 4EBP-1 can be inhibited by Rapamycin which is a special inhibitor of mTOR. Correspondingly the phosphorylation of S6K1 and 4EBP-1 decreased when mag-1 expression was inhibited by siRNA. A typical mTOR activation signal pathway is PI3K/Akt/mTOR/S6K1(4EBP-1). Therefore we also tested the effect of mag-1 on the phosphorylation of Akt and found the alteration of mag-1 expression level could not cause the phosphorylation of Akt suggesting that mag-1 promoting S6K1 and 4EBP-1 phosphorylation probably lie downstream of PI3K/Akt/mTOR/S6K1(4EBP-1) pathway.In one hand mag-1 enhances tumor metastasis, in another hand it induces mTOR pathway activation, which suggested mag-1 may promote tumor metastasis through activating mTOR signal pathway. In order to test the hypothesis, we examined the behavior of tumor cells treated with Rapamycin and found that Rapamycin reduced the enhanced migration, adhesion and invasion resulting from mag-1 overexpression.In summary, there is a high correlation between mag-1 and tumor metastasis. The activation of the mTOR pathway induced by mag-1 overexpression plays an essential role in the increased migration, adhesion and invasion of cells in response to mag-1.