Effects of heparanase gene silencing by miRNA interference on invasion and migration of gallbladder carcinom cells

Objective To investigate the effects of heparanase (HPA) gene silencing by miRNA interference on invasion and migration of GBC-SD cells.Methods Recombinant plasmid four pairs of single-stranded miRNA oligo-DNA and a pair of negative control plasmid expression vector targeting human HPA gene sequence were respectively transfected into GBC-SD cells by LipofectamineTM 2000,Reverse transcription-polymerase chain reaction (RT-PCR) were used to detect the mRNA expression changes of HPA before and after transfection and adopt to select the recombinant plasmid which showed the best silencing effect.Stable transfectants GBC-SD cells can be selected using 2 mg/L antibiotics Blasticidin pressurescreening and positive clones for further experiments.Matrigel invasion assays and wound closrue assays were performed to examine the migration and invasive ability of the GBC-SD cells.Results Recombinant plasmid pcDNA6.2-GW/EmGFP-miR-HPA-2 is the best one to silence mRNA expressions of HPA.Howerer,level of recombinant plasmid pcDNA6.2-GW/EmGFP-miR-HPA-2 group GBC-SD cells is the most decreased by transwell invasion assay,compared with the negative control and blank,the difference was significant (P ＜ 0.05).GBC-SD cells of recombinant plasmid pcDNA6.2-GW/EmGFP-miR-HPA-2 group migration distance at 0,12,24 h was the most lowest compared the negative control and blank,the difference was significant ( P ＜ 0.05).Conclusion HPA mRNA of hunan gallbladder carcinoma GBC-SD cells are silenced by miRNA interference. ( Invasion and migration of human gallbladder carcinoma GBC-SD cells are inhibited afterHPA gene is silenced.) The recombinant plasmid pcDNA6.2-GW/EmGFP-miR can effectively suppress HPA mRNA expression,inhibit cellular invasion and migration in GBC-SD cells in vitro. Key words: Gallbladder carcinoma;  Gene silencing;  miRNA interference;  Heparanase; Tumor invasion