| Background and Objective:Colorectal cancer is one of the malignant cancer threatening human healthy. There is still no effective cure for colorectal cancer. The traditional operation has curative effects slowly and large wound to patient, so in recent years, survival rate of colorectal cancer patients had no significant increase as the development of tumors. Molecular biological methods are the hopeful way for the therapy of the patients. The gene therapy targeting to malignant tumor is hopeful to turn over a new page.The RNAi technology provides basis for gene therapy of tumors. So, the choice of a good target site is the key for gene therapy. Tumorgenesis involves a large amount of interference ogene, involving more than a vast complex regulatory network. Lef-1 gene locates on human chromosome 4q23-4q25. The size of mRNA reading open frame (ORF) is 1200 bp, the genome sequence spans at least 140 kb which contains 12 exons and 11 introns. The third intron may contain an alternative exon. Lef-1 is a multi promoter gene, the first promoter, transcribes a full length mRNA, and encoding a product contains beta-caten biding area. The second promoter transcrip a truncated form of Lef-1 and lacks a beta-catenin binding area, which has a negative control of wnt signal transduction. LEF-1 is closely relevant to tumorgenesis, tumor progression and invasion. As a member of TCF/LEF family, Lef-1 finishes Wnt pathway through recruiting transcription factors to target gene. The oncogenesis caused by abberant Wnt pathway is based on theβ-catenin accumulated in cytosol. The streaming overβ-catenin translocates into the nucleus and binds to TCF/LEF, activates the transcription of the down stream target gene c-myc,yclin-D1,gastrin,survin,VEGF,ASEF leading to tumorgenesis. Therefore, block the abmormal wnt signals can restrain tumor cell proliferation, and induce apoptosis. LEF/TCFs-β-catenin as the ultimate effector of wnt signal transduction will be an important target for anticancer treatment. Targeting the LEF/TCF binding sites onβ-catenin to develop small-molecule drugs closed unique signal molecules that are effective strategies for the cure of tumor at molecular level.With technical innovation, people coming to find a lot of new genes, so there are more methods to study gene function. RNAi becomes an effective tool to study specific gene. It has great prospect in the field of molecular area.RNAi has the advantages of operating convenience, cheap, efficient, so it can be used in almost all of the gene sequence. Therefore, RNA interference has potential and broad prospects in cancer diagnosis and treatment.This paper based on the RNAi study at home and abroad, directed at three variants of LEF-1 gene, and design three interference targeting the gene sequence. Using Caco-2 cells as the target cells, the RNAi inhibition efficiency was confirmed by using western blot. The LEF-1 function was evaluated by using hoechst 33258 staining, MTT and wound enclosure methods.Method:1. Search for human LEF-1 gene sequence of each mRNA variant people in GenBank, accoding to RNAi design principle, design three interference targeting LEF-1 missense sequence and a negative control sequence.2. Grope a new reagent S-TranG which greatly improved transfection efficiency. Group (NC, siRNASCR, siLEF1-1, siLEFl-2, siLEFl-3), and import plasmid into CacO-2 cells.3. Western blot to detect LEF-1 protein expression level at 24h,48h,72h,96h post transfection.4. Using MTT, Hoechst 33258 staining and wound enclosure in vitro method to detect changes in proliferation, migration and apoptosis morphplogy after RNA interference.Result:1. The enzymes cutting and DNA sequence testing confirmed the build of LEF-1 siRNA plasmid is a successful.2. Grope a new reagent S-TranG which greatly improved transfection efficiency (cell coverage is about 60%-70%, plasmid dosage is 4μg, quiencing is 10 to 15 minutes).3. The results of western blotting showed that:the expression level of group siLEF1-3 decreased,restained effects is the highest at 72h post transfection.4. The results Mtt, hochast33258 staining and damage repair in vitro method show that the tree interfere groups all have restain effects on Caco-2 cells.Cell migration and proliferation is obviously restained (p<0.01). Hochast332581 staining indicates that there is apoptosis feature in interference goup, and there is no obviously difference in proliferation, migration and apoptosis morphplogy compared with contrl negative group.Conclusion:RNA interference technology aims to silence LEF-1 gene expression in human colorectal cancer cells (caco-2) and to hold the cell proliferation and migration ability, promote cell apoptosis. There is no substantial change in proliferation, apoptosis and migration rate in negative control group. Western blotting shows that the protein level significantly reduced in interference group. So, LEF-1 is hopeful to become a new target for colorectal cancer treatment, and provides new strategies for colorectal cancer treatment. LEF-1 gene intervention will have a huge application potentiality in future. |
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