Construction of miR-200c gene recombinant lentiviral vector and establishment of multi-drug resistance animal models in human colon cancer

LIU Chong; SUI Hua; ZHU Hui-rong; LI Dong; LI Qi

doi:10.3969/j.issn.1002-266X.2015.20.004

Abstract：Objective To construct the lentiviral vector co-expressing human miR-200c gene and luciferase , and es-tablish a stable colorectal cancer multidrug resistance ( MDR) cell model , in order to lay an experiment foundation for ex-ploring the function of miR-200c in MDR.Methods miR-200c gene was cloned into the lentiviral vector (pGC-Lentivir-us), which was verified by PCR and sequencing analysis .After sequencing, we made lentiviral vector and titer test .Using cell transfection technique , miR-200c lentiviral vector was infected into MDR cell line HCT-116/L-OHP.A nude mouse model of colorectal MDR cancer was established by subcutaneous inoculation of this kind of cell ; meanwhile , the empty vector was taken as the control group .Experimental nude mice were randomly divided into 4 groups after 2-week tumor-growth.Mice in the control group were administered with normal saline daily .Mice in the experiment group were adminis-tered with oxaliplatin (L-OHP).After treatment for 4 weeks, mice in each group were sacrificed to measure tumor volume . Results The best titer was 2 ×108 TU/mL.The qPCR showed that the levels of miR-200c mRNA expression in the resist-ant cells of human colon cancer which were transfected by miR-200c-Lentivirus were significantly higher than those in the empty vector group and the blank control group ( all P<0.01).In vivo imaging confirmed that HCT-116/L-OHP-miR-200c-luc cells with luciferase could be tested quickly and sensitively , and could be used to watch the tumor growth .Com-pared with HCT-116/L-OHP-luc group, HCT-116/L-OHP-miR-200c-luc group showed more sensitive to L-OHP ( P <0.05).Conclusion The lentiviral screening vector with human miR-200c gene is successfully constructed , which realizes the stable integration of the recombinant in the human colon cancer HCT-116/L-OHP MDR cells;meanwhile, its MDR co-lon cancer animal models which have miR-200c overexpression with luciferase are successfully constructed .