Objective:To explore the effect of CXCR7shRNA lentiviral vector on CXCR7 expression of human colon cancer cell line HT-29.
Methods :Three siRNA interference sequences targeting CXCR7 gene were designed. Double-stranded shRNA hairpins were synthetized and separately cloned into plVTHM vector digested by double-enzyme so as to recombinate 3 shuttle-plasmids of plVTHM shRNA1, plVTHM shRNA2, plVTHM shRNA3. Then these recombinant plasmids were transformed into DH5α competent cells. After Amp screening positive clones, plasmid was taken from line for subsequent identification by enzyme-digestion and DNA sequencing analysis. The titer of lentivirus was determined after 293T cells were cotransfected with plVTHM shRNA and lentiviral packaging plasmids. The 3 kinds of recombinant lentiviruses and negative control lentiviruses were used to infect HT-29 cells and then CXCR7mRNA and protein were detected by real-time PCR and Western blotting, respectively.
Results:Identification of enzyme-digestion and DNA sequencing analysis confirmed that the 3 CXCR7shRNA sequences were successfully inserted into the lentiviral vectors with the titers of concentrated virus 6×107TU/mL, 4×107TU/mL and 5×107TU/mL, respectively. CXCR7 expression in HT-29 cells was significantly decreased at both mRNA and protein levels compared with the non-transfected and negative control vector (P<0.05). The LV-CXCR7shRNA-2 and LV-CXCR7shRNA-3 played a significant role in reducing mRNA by 72% and 67%, protein by 68% and 55% (P<0.05), respectively; the silent effect showed no significant difference between the nontransfected and negative control vector (P>0.05).
Conclusions:The 3 lentiviral shRNA expression vectors targeting CXCR7 gene were successfully constructed and can effectively downregulate the expression of CXCR7 in HT-29 cells.Thus it provid stable cell transfection vector for further study of CXCR7 gene-targeted gene therapy on colon cancer