| BACKGROUND:At present, breast cancer incidence of a disease has grown women the malignant tumor, and transfer is lead to breast cancer patients the major causes of death. Distant metastasis happens patients with breast cancer, the5-year survival rate is only23%. Therefore, to further explore the invasion and metastasis of breast cancer related mechanism, looking for new treatment method, improve breast cancer treatment effect, improve breast cancer survival rates are important. Recently, found on lysyl oxidase (LOX) in the affected the expression in metastatic breast cancer increases, and LOX high expression of breast cancer patients without distant metastases surial and oerall surial is relatively short. Lysyl oxidase(LOX) is the key enzyme,the Extracellular matrix collagen and elastin polymerization and stable at the start of the Extracellular matrix (ECM), it can promote collagen and elastin happened between crosslinking, thus increasing the stability of the Extracellular matrix, and at the same time, LOX also has the function of adjusting the cell adhesion, sports and gene transcription etc. The expression of LOX has significant differences in different tumor tissue and cell lines. Research found that the expression of LOXmRNA only in high hit/transfer breast cancer cell lines MDA-MB-231, and low hit/transfer breast cancer cell lines in MCF-7without expression. Pointing out that LOX plays a very important role in breast cancer invasion and metastasis of process. Now thinking that, in breast cancer LOX is likely as tumor metastasis promote factors plays an important role of biology.OBJECTIVE:To construct LOX RNA interference slow virus carrier and packing, and then transfect stably to breast cancer cell lines MDA-MB-231, and then determine the interference efficiency, research on the expression of the breast cancer related factors MMP-2, MMP-9, HIF-1alpha, Ki-67, CyclinDl mRNA after suppress LOX gene expression, and to investigate the influence of RNAi against lysyl oxidase (LOX) gene on growth, migration and invasion. To explore the possible mechanism of LOX in invasion and metastasis of breast cancer.METHODS:1. Breast cancer cell lines MDA-MB-231bought in Shanghai cell bank of Chinese academy of sciences, is good cell lines after subculture cell. Set up three experimental groups, namely blank group (Con), interference group (RNAi), negative control group (Mock),and then to interfere, after interference using Real-time PCR and Western-blot to test the interference efficiency of LOX mRNA and protein expression, and application related statistical methods are analyzed.2. Lentiviral interference vector (LOX-RNAi-LV) was transfected into the breast cancer cell line MDA-MB-231. The expressions of LOX mRNA, MMP-2mRNA, MMP-9mRNA, HIF-1αmRNA, Ki-67mRNA and CyclinD1mRNA were detected by Real Time PCR, and detected the expression of LOX protein by the Western blot, then application related statistical methods are analyzed.3. The breast cancer cells were divided into three groups:the experimental group (RNAi), the negative control group (Mock) and the blank control group (Con). Lentiviral interference vector (LOX-RNAi-LV) was transfected into the breast cancer cell line MDA-MB-231. The expressions of LOX mRNA was detected by Real Time PCR, and the expression of LOX protein was detected by Western blot. The abilities of growth and proliferation the transfected cells were determined by MTT assay, cell colony formation and Flow cytometric. The abilities of migration and invasion were observed by cell invasion and motility test (Transwell). And application related statistical methods are analyzed.RESULTS:1. After stability transfect slow virus RNAi, LOX mRNA and protein expression are suppressed, compared with blank group, inhibition rate of LOXmRNA in RNAi group was89.2%; inhibition rate of LOX protein in RNAi group was82.97%; differences are significant.2. After stability transfect slow virus RNAi, Real-time PCR results in Con group, RNAi group and Mock group show that MMP-2gene expression of mRNA were1.000±0.000,0.496±0.021and0.846±0.047; MMP-9gene expression of mRNA were1.000±0.000,0.571±0.099and0.786±0.042; HIF-1a gene expression of mRNA were1.000±0.000,0.492±0.003and0.780±0.041; Ki-67gene expression of mRNA were1.000±0.000,0.588±0.036and0.942±0.050; CyclinD1gene expression of mRNA were1.000±0.000、0.411±0.014and0.762±0.034, F were225.271,35.373,341.081,117.561and604.828, P<0.01, above the mRNA gene expression in the interference groups decreased, in different groups expression differences with a statistical significance.3. After stability transfect slow virus RNAi, in the MDA-MB-231cell line, LOX mRNA and protein expression level are reduced and the cell growth was significantly inhibited, cell JiLa formation rate reduced significantly, the average cells JiLa formation rate of the RNAi group, Con group, Mock group are (15.67±2.082)%、(30.67±2.082)%and (27.67±2.082)%, RNAi group of cells JiLa formation rate was significantly lower than the Con group and Mock group, and JiLa cells are obviously reduced the number of control group. Cells invade ability are significant inhibited, RNAi group, Con group, Mock group through Transwell small room average filtration membrane cell number47±2、100±1、88±2; Cell migration ability are significant inhibited, RNAi group, Con group, Mock group through Transwell small room average filtration membrane cell count to63±2、118±2、96±2.Through the Transwell RNAi group small room filtration membrane cells were significantly less than Con group number and Mock group, a statistically significant difference (P<0.05); Flow cytometric determination of the cell cycle, the difference between the three groups was statistically significant.CONCLUSION:1. Slow virus mediated RNA interference has the characteristic of high efficiency, stable, strong specific; and can generate siRNA in a long time and stably in cells to realize the stable of RNA interference.2. Transfect LOX-RNAi-LV can effectively cut the LOX mRNA and protein expression level of breast cancer cells MDA-MB-231.3. After the LOX gene expression restrained,the ability of proliferation and the invade and the migrate of MDA-MB-231breast cancer are suppressed.4. The mechanism that LOX promote breast cancer growth proliferation may be related to the abnormal expression of factor Ki-67and CyclinD1; after restrain the LOX gene, the expression of Ki-67and CyclinD1have significantly reduced. 5. The mechanism that LOX promote breast cancer invasion and metastasis may be related to the abnormal expression of factor MMP-2、 MMP-9and HIF-1α; after restrain the LOX gene, the expression of MMP-2、MMP-9and HIF-1α have significantly reduced. |
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