Screen And Identify The Potential Downstream Target Genes Of FOXQ1 In Colorectal Cancer By Using Microarray And Luciferase Reporter System

Chacpter One Microarray analysis provides new insights into the function of FOXQ1 in colorectal cancerAIM:To compare the difference of whole genomics expression before and after knocking down FOXQ1 in human colorectal cancer cell line DLD1.METHODS:After extraction RNA from DLD1 cell lines, hybridize with the probes on Whole Human Genome Oligo Microarray (4x44K, Agilent Technologies) and miRCURYTM LNA Array (v.18.0, Agilent Technologies). Mmultiple target genes were then analyzed by GeneSpring and Genepix software. Quantitative Real time PCR was applied for the validation of microarray. The statistical analyze was applied by Microsoft EXCEL and SPSS 17.0.RESULT:Make｜ FC｜>2 as the significant difference standard, with the gene FOXQ1 knocking down, in 41093 tested genes,255 were up-regulated and the other 176 were down-regulated. The result was also certificated by qRT-PCR. Make｜ FC｜>2 as the significant difference standard, with the gene FOXQ1 knocking down, in 2075 tested MicroRNAs,31 were up-regulated and the other 12 were down-regulated. It proves FOXQ1 influences the cytokine-cytokine receptor pathway. We also found microRNA-208b-3p and microRNA-320d as the potential upstream of FOXQ1, which probably knock down FOXQ1 expression in colonal cancer cell line DLD1.CONCLUSION:The whole genome microarray provides clues and evidence for FOXQl’s function in the development and progression of colorectal cancer.Chacpter Two Undertake the direct transcriptional activation genes of F0XQ1 using dual luciferase in colon cancerAIM:In order to find out the direct transcriptional activation of F0XQ1 in human colon cancer cell line DLD1.METHODS:By using the tools of bioinformatics Jaspar of FOXQ1 target sites prediction, of HBEGF, PDGF beta, PDGFR, PLAT, IL1 beta contains 5 gene promoter region of firefly luciferase plasmid PGL3-enchancer construct. Then, co transfect PGL3 plasmid with PRL-TK and shFOXQ1-DLD1 cells shcontrol-DLD1, using luciferase reporter assay system to detect the fluorescence intensity.RESULT:Successfully got the five luciferase plasmids contain HBEGF, PDGF beta, PDGFR, PLAT, IL1 beta 5 gene promoters, and proved that FOXQ1 in colorectal cancer cell DLD1 cannot directly activate the transcription of HBEGF, PDGF beta, PDGFR, plat and IL-lbeta gene.CONCLUSION:The experiment eliminates the direct target genes of FOXQ1 in colorectal cancer cell DLD1.