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Effects of CPB2 Gene Silencing on TAFI Expression and Metastatic in the Breast Cancer Cells

Author: WangZeZuo
Tutor: XuChengWei
School: Shandong University
Course: Clinical Laboratory Science
Keywords: Breast cancer RNA interference (RNAi) Thrombin - activatable fibrinolysis inhibitor ( TAFI )
CLC: R737.9
Type: Master's thesis
Year: 2011
Downloads: 33
Quote: 0
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Abstract


The purpose of breast cancer is one of the most common malignancy in women, seriously endanger the lives and health of patients. Patients can occur in early cancer lesions transfer is not conducive to the effective control and treatment of breast cancer, anti-metastatic breast cancer treatment to become a research hotspot. Thrombin-activatable fibrinolysis inhibitor (TAFI) is a newly discovered secreted by the liver plasma carboxypeptidase-like activity of single-chain glycoprotein. The TAFI thrombin, plasmin and thrombin - thrombomodulin protein complex (T-Tm) to activate the formation of activated TAFI (TAFIa), TAFIa can degrade the fibers of partially hydrolyzed protein C-terminal lysine residue, inhibit plasminogen activation, high concentrations of TAFIa can also directly inhibit the activity of plasminogen, a further decline in the activity of the fibrinolytic system, has close ties with clinical heart, cerebrovascular disease, cancer and many other diseases. The TAFI concentration coding genes carboxypeptidase B2 (CPb2) control, CPB2 positioning at 13q14.11, the TAFI mRNA translation synthetic protein consisting of 423 amino acids, including a signal peptide of 22 amino acids, 92 amino acids of the active peptide and The activity of the catalytic domain of 309 amino acids. MDA-MB-231 breast cancer cell lines using RNA interference technology, through the design of small interfering RNA (siRNA), to targeted inhibition of gene expression in breast cancer MDA-MB-231 cell lines CPB2 RNA interference is detected in breast cancer cells TAFI expression levels; TAFI and to explore the impact on breast cancer cell growth and metastasis; study the relationship between tumor and blood coagulation, fibrinolytic system; combined study further found that the Cancer Therapy ideas. Method 1. Recovery, cultured breast cancer MDA-MB-231 cells Department of passage to logarithmic phase; 2 3 candidate-specific siRNA design and chemical synthesis coding TAFI gene (CPb2), and a non-specific siRNA, the The cells were divided into siRNA-1, siRNA-2 group, siRNA-3 group, negative control group and blank control group of five groups, using Lipofectamine 2000 transfection of MDA-MB-231 cells by RT-PCR detection; 3. TAFI mRNA expression in MDA-MB-231 cells after transfection; Western Blot method to detect the transfection TAFI protein expression in MDA-MB-231 cells; After RT-PCR and Western blot The experimental results filter out the highest inhibition rate of siRNA further Transwell invasion assay after transfection of MDA-MB-231 cells invasion and metastasis of capacity, compared with negative control group and blank control group; 6-methyl thiazolyl tetrazolium (MTT) assay after transfection of MDA-MB-231 cell survival and growth, compared with negative control group and blank control group. 1 cells divided into five groups successfully transfected, RT-PCR testing found that siRNA-1 group, siRNA-2 group, siRNA-3 group and negative control group comparison, the TAFI mRNA expression level was significantly decreased; 2. Western Blot detection 5 groups of cells TAFI protein expression amount, found that siRNA-1 group, siRNA-2, siRNA-3 group compared with the negative control group, the TAFI protein expression level was significantly decreased, the three siRNA transfer dye group of inhibition rate was (55.4 ± 4.9)%, (55.0 ± 0.6)%, (42.6 ± 3.5)%; 3RT-PCR and VWestern, blot screening of siRNA-3 siRNA highest inhibition rate of the Transwell invasion was found the group after transfection, cells invasion and metastasis capacity than the negative control group was significantly lower; 4.MTT experiment TAFI expression by inhibiting siRNA-3 group cell growth was inhibited, reduced survival. Conclusion The success of RNAi technology to inhibit the expression of TAFI in breast cancer MDA-MB-231 cell lines, so that the breast cancer cell TAFI mRNA and TAFI protein content was significantly reduced, especially the most significant inhibitory effect of siRNA-3 group, and therefore screened siRNA -3 further study TAFI growth and metastasis of breast cancer cells. TAFI level reduced to the activity and invasion of breast cancer cells was significantly reduced. TAFI inhibition of fibrinolysis thereby reducing the activity of tumor cells and the invasion force, has a very close relationship with tumor progression and metastasis, but also provides a new approach to cancer treatment.

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CLC: > Medicine, health > Oncology > Genitourinary tumors > Breast tumor
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