Construction Of MiRNA Expression Vector Of Human GRP78 Gene And The Effect On Proliferation Of EC109 Cells

Objective:To construct an effective miRNA expression vector of human GRP78 gene and to observe its effect on proliferation of EC109 cells.Materials And Methods:1. Four oligonucleotides of human GRP78 were cloned into pcDNA^TM6.2-GW/EmGFP-miR expression vector, which was named miR78-1, miR78-2, miR78-3, miR78-4 respectively. The four recombiant vectors were identified by sequencing.2. The four recombiant vectors and a negative control vector were transfected into HEK293 cells using Lipofectamine 2000. The green fluoresent proteins (EGFP) were measured by fluoresence microscopy in cells to observe transfection efficiency. After treatment with blasticidin in two weeks, the stable transfectants cells with GRP78-miRNA were obtained. Reverse transcriptional PCR(RT-PCR) was performed to evaluate the effects of gene silencing.3. The best interferential plasmid (GRP78 miRNA) was selected and transfected into EC109 cells. 24 h after transfection, expressions of GRP78 mRNA were detected by RT-PCR. The proliferation of EC109 cells was determined by CCK-8 assay in 0, 12, 24, 48, 72 h after transfection.Results:1. All recombiant vectors were successfully constructed by sequencing and no any gene was lost or mutated. 2. EGFP were observed in all recombiant HEK293 cell lines. Expressions of GRP78 mRNA of HEK293 cells transfected with GRP78-miRNA were decreased (all P<0.05), compared to the untransfected group or negative control group, and the most decrease was observed in miR78-1 (P<0.05) group.3. 24 h after transfection, expressions of GRP78 mRNA in EC109 cells decreased (P<0.05), compared to the untransfected group or negative control group. EC109 cell proliferation were suppressed significantly after transfection in 12, 24, 48, 72 h (P<0.05), compared to untransfected group.Conclusions:1. All recombiant vectors of human GRP78 were successfully constructed and identified.2. The stable transfectants of GRP78 knockdown in HEK293 cells with microRNA were obtained, and the best gene silencing efficiency vector (miR78-1) was selected.3. After transfection with miR78-1 vector, the expression of GRP78 mRNA was obviously inhibited in EC109 cells, and that was sufficient to inhibit the proliferation of EC109 cells.