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Background and Purpose of colorectal cancer is a malignant tumor of harm to human health. Has recently witnessed a year-on-year growth trend. Treatment of colorectal cancer is currently no effective treatment, the traditional surgical treatment has efficacy of slow, trauma and other defects, resulting not significantly improve survival in patients with colorectal cancer in recent years. The development of molecular biology brings hope for the treatment of tumors. Tumor gene as a target for gene therapy is expected to turn a new page for the treatment of cancer and other malignant diseases. Can use the current rise of RNA interference for the study of the function of tumor-related genes, targeted gene therapy research. Therefore, choose a good target for gene will be the key to gene therapy. Tumorigenesis involving multiple genes, is a large and complex regulatory networks. LEF-1 gene is located on human chromosome 4q23-4q25 size of 1200 bp mRNA open reading frame (ORF), its genome sequence at least span of 140 kb, contains 12 exons and 11 introns, which has a large 3 (75 kb) of intron may contain optional exon. LEF-1 is more than one promoter gene, the first startup of LEF-1 mRNA transcription full length, encodes a protein having a product of the binding region of β-catenin, second promoter transcription truncated form of LEF-1, absence of β -catenin binding domain, has negative regulation of the Wnt signal. Tumor development, invasion and metastasis. LEF-1 as one of the LEF / TCF family members by raising transcription activating factor β-catenin target genes mediated Wnt signaling pathway. The Wnt carcinogenic β-catenin can not be degraded, the excessive intracellular β-catenin into the nucleus and with LEF / TCF combined activation of downstream target genes c-myc, cyclin-D1 astrin, survin, VEGF, ASEF's transcription , resulting in tumorigenesis. Therefore, blocking the abnormal Wnt signaling pathway can inhibit tumor cell proliferation and induce apoptosis of tumor cells. LEF / TCFs-β-catenin as the end-effector of the Wnt signaling pathway, will be an important target for anti-cancer treatment. The development of small-molecule drugs for the β-catenin LEF / TCF binding sites closed to the specific signal molecules, molecular level treatment of tumors strategy. With the innovation of technology, people have been discovered many new genes, more and more research methods on gene function, RNAi effects tools to study specific genes, has a great prospect in the field of molecular. RNAi agile operation, Chen, efficient, short cycle, the advantages of high specificity, can be applied to almost all of the gene sequence. Therefore, RNA interference technology in the diagnosis and treatment of tumors with potentially broad prospects. The paper at home and abroad on the basis of RNAi research design targeted for LEF-1 gene three variants the three interference sequence of the gene to colorectal cancer Caco-2 cell lines as experimental material, the use of RT-PCR Western blot, Hoechst33258 staining, MTT assay and in vitro damage repair method and other methods to study the LEF-1 gene was interfering with mRNA levels, changes in the level of protein expression, apoptosis morphological changes, proliferation and migration of change . Method 1. Search people LEF-1 mRNA sequence of the gene variant, in accordance with the principle of siRNA design, design optimization of the three targeted interference of LEF-1 sequence and a negative missense control sequences in GenBank. The synthesis of these sequences, the sequences cloned into vector Pu6.1-GFP. Explored new transfection reagent S-TranG greatly improve the efficiency of transfection group (NC, shRNASCR, shLEF1-1, shLEF1-2, shLEF1-3) and the plasmid was introduced into human colorectal cancer (Caco- 2) cell lines; transfection were detected by western blot 24h, 48h, 72h, 96h after each group LEF-1 protein levels; MTT Hochast33258 staining and damage repair in vitro were observed by analysis to suppress the expression of LEF-1 (Caco-2) after human colorectal cancer cell proliferation and apoptotic morphological changes in vitro and athletic ability. Results 1. Confirmed by restriction enzyme digestion and DNA sequencing LEF-1 siRNA recombinant plasmid vector was constructed successfully. Explored new transfection reagent S-TranG greatly improve the efficiency of transfection (transfection rate of 60% -70% confluence 4μg plasmid dosage and let stand for 10-15 minutes) recombinants efficient to import human colorectal cancer cells. Western blotting results show: the the interference group after transfection of LEF-1 protein levels 72 hours after transfection protein inhibition rate compared to other interference group and siLEF1-3 inhibition inhibition efficiency more. 4.MTT law, the Hochast33258 staining and damage repair in vitro experimental results show that the three interference groups of Caco-2 cells have inhibitory effects, 72h, 96h cell proliferation and migration was significantly inhibited (p <0.01), Hochast33258 staining showed transfection 96 h, the interference group observed characteristics of apoptosis cells, negative control group, the proliferation, apoptosis and migration after transfection with the normal control group had no significant difference in conclusions by RNA interference target to silence the LEF-1 gene in the negative control group, the proliferation, apoptosis and migration without colorectal cancer expression in Caco-2 cells, inhibits Caco-2 cells in vitro proliferation and migration, to promote Caco-2 apoptosis; significant changes. The interference group by Western blot analysis of protein expression levels were significantly lower. Thus, LEF-1 is expected to become a new target for the treatment of colorectal cancer, and provide a new basis for the treatment of colorectal cancer and strategies, will have great potential for application of the intervention of the LEF-1 gene expression.
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