Rna Interference Ard1 Colorectal Cancer Cell Line Cell Cycle

Colorectal cancer (CRC) is a common malign tumor in digestive duct.Recently, due to diet changes and various living styles, CRC rate keeps increasing, especially in highly-developed areas. CRC is presently considered as a multi-factors, multi-genes and multi-steps developed disease. With a further understanding of molecular biology, CRC researches are developing more microscopically, and target therapy is a focus of attention.Recent research shows that ARD1 is related to breast carcinoma, thyroid cancer and lung cancer. And recent study reveals a cDNA Tag consequence AAGCCGGACC in breast cancer, of which expression level is depressed after tamoxifen treatment. In research of H1299 and A549 in lung cancer cell line, after hARD1 silence of RNAi, cell proliferation is reported to be depressed, and rest on G1 phase; after hARDl knockout, cyclin D1 promoter is depressed, meanwhile beta-catenin/TCF which related to cyclin D1 is depressed too. Cyclin D1 has already been proved of a oncogene, which limits G1/S phase of cell proliferation. But it is worth researching that whether cyclin D1 is also depressed in colorectal carcinoma as that in lung cancer, and that it provides new reference for research of ARD1 in colorectal carcinoma.Gene silence by antisense oligonucleotide transcription, is widely adopted in studies of gene function, gene controlled expression mechanism and many fields, and what is more important is that RNAi establishes a firm foundation for gene therapy. As further research of ARD1 proceeds, its importance to cell differentiation, proliferation and stability makes study of ARD1 molecule biology function in highlight. As discussed above, it will be a interesting experiment of ARD1 by RNAi. In this experiment, we operate a silence transfection of ARD1 in colorectal carcinoma cell lines, then analyze cell phase by flow cytometry. Aim to confirm ARD1 function to cell phase.Objective:Screen effective silence siRNA based on ARD1 gene consequence. Study cell phase changes of colorectal carcinoma after ARD1 silence to SW620, LS-174T and HCT-8. And try to reveal influence of ARD1 to colorectal carcinoma cell phase.Methods:1. SW620, LS-174T and HCT-8 preparation.2. Transfect these three cells by 7 siRNA.3. Analyze experiment group and control group by high content imaging system.4. Extract RNA by Trizol method, and count total RNA.5. Extract cDNA following Promega reverse transcript kit.6. Screen siRNAs which have effective silence function by PCR.7. Transfect colorectal carcinoma cells by effectual siRNA.8. Examine cell phase by flow cytometry.Results:1. Transfection rates of siRNA are all above 70%.2. Screen out 4 siRNA, target sequences are CAGAGGACCTAATGAACAT, GGTGGAGAGCAAAGGCAAT CCATGATAGAGAACTTCAA, GAGCTTTCACAATAAATTT. Silence rates are 76.6%,79.7%,74.3% and 75.8% respectively.3. Perform relatively quantitative analysis byΔΔCT to analyze siRNA, using P-actin and GAPDH. Results are basically the same.4. After transfection, count DNA content of G0/G1 by flow cytometry. DNA content of SW620 is 52.91,50.93 and 17.81 in 24h,48h and 72h:DNA content of LS-174T is 55.92,55.3 and 20.35 in 24h,48h and 72h:DNA content of SW620 is 39.57,40.22 and 44.17 in 24h,48h and 72h.5. After transfection, count DNA content of G2/M by flow cytometry. DNA content of SW620 is 1.24,5.42 and 23.82 in 24h,48h and 72h:DNA content of LS-174T is 5.63,1.19 and 42.7 in 24h,48h and 72h:DNA content of SW620 is 4.22,15.64 and 19.55 in 24h,48h and 72h.6. Compared experiment group with control group, in G0/G1 phase, DNA content of all three cells fall; in G2/M phase, DNA content arise, with statistic difference.Conclusion:Cell phases of SW620, LS-174T and HCT-8 rest on G0/G1 phase after ARD1 transfection, and G2/M activity arises.