Constrution Of FOXQ1-silenced Colorectal Cancer Cell And The Research Of Relationship Between FOXQ1 And EGFR Gene

Part Ⅰ:Construction of lentivirus expression vector and to silence the expression of FOXQ1 gene in colorectal cancer cell line DLD-1Objective:To construt and identify the lentivirus vectors and silence the expression of FOXQ1 gene in colorectal cancer cell line DLD-1.Methods:According to FOXQ1 gene sequence, three pairs of shRNA were designed and synthesized then ligated to the lentivirus PLKO.1 vector. After restructured plasmid was transfected into STB 13 competence cells and cultivated on the amicillin resistance culture plate, single colony was picked to be enlarged cultivation, then restructured plasmid was extracted.lt was extracted without endotoxin that the restructured plasmid identified by enzyme digesting and sequencing. Restructured plasmid and auxiliary packaging plasmid including pRSV-rev, pMDlg-pRRE, pCMV-VSV-G were jointly infected into 293-T cells by calcium phosphate tranfecting method, then the lentivirus supernatant was obtained and used for infecting DLD-1 cells.After selection with puromycin, the FOXQ1-silent cells were obtained, then then efficiency of gene knockdown were determined by Real-time PCR and western blot.Results:Enzyme digesting and sequencing results showed that shRNA were successfully inserted into PLKO.1. Recombinant plasmid was transfected into 293-T cells and high-titer lentivirus was formed. The lentivirus was transducted into DLD-1 cells. FOXQ1-silent cells were obtained after seletion. Real-time PCR data and western blot showed the highest inhibitory efficiency was approximately 90.4% for FOXQ1 expression.Conclusion:FOXQ1-silent cells were successfully obtained by construting lentivrius interference system. It provid an experimental foundation for further researching of function of FOXQ1 gene in tumorigenesis.Part Ⅱ:The research of relationship between FOXQl and EGFR geneObjective:To discuss the relationship between FOXQ1 and EGFR gene in colorectal cancer.Methods:To detect the mRNA relative expression quantity of FOXQ1 and EGFR gene in colorectal cancer cell lines by Real-time PCR; To detect the mRNA relation expression quantity of EGFR in DLD-1 cell which was interfered by lentivirus (named DLD1-shRNA-FOXQ1); After DLD1-shRNA-FOXQ1 was processed by EGFR tyrosine kinase inhibitor Erlotinib HCl and siRNA-EGFR,We detect the mRNA relative expression quantity of FOXQ1 and EGFR gene by Real-time PCR.Results:(1)Expression levels of FOXQl and EGFR gene in colorectal cancer cell lines:Respectively relative expression quantity of FOXQ1 in DLD-1, HT29, HCT116, LOVO were 83.09,59.58,0.03,0.06; Respectively relative expression quantity of EGFR in DLD-1, HT29, HCT116, LOVO were 4.95,3.67,1.36,2.08; (2)EGFR relative expression quantity in DLD-1 cell which was interfered by shRNA-FOXQ1 was increased with decrease of FOXQ1; (3)After DLD1-shRNA-FOXQ1、 DLD1-shRNA-Cr and DLD-1 were respectively processed by Erlotinib HCl and siRNA-EGFR, FOXQ1 relative expression quantity was increased with decrease of EGFR.Conclusion:FOXQ1 and EGFR gene expression trend was consistent in colorectal cancer cell lines. There was negative relationship between FOXQ1 and EGFR in colorectal cancer. That may exist negative feedback regulation mechanism between the two, thus maintaining high expression levels of FOXQ1 and EGFR in colorectal cancer cell.