Influence Of Microrna Silencing ILK Gene On Proliferation Of Human Bladder Tumor BIU-87 Cells

Objective: To study influence of miRNA silencing ILK gene on proliferation of human bladder cancer BIU-87 cells,through constructing bladder cancer BIU-87 cells strain that stably expressed the specific ILK miRNA.Methods:1. Four ILK specific interference sequences and a negative control sequence targeting on human ILK were designed respectively, and miRNA interference vectors: pcDNA^TM6.2-GW/EmGFP-miR-ILK-1, 2, 3, 4 (miR-ILK-1, 2, 3, 4) and pcDNA^TM6.2-GW/EmGFP-miR-ILK-neg (miR-ILK-neg), were constructed. All of these were transfected into BIU-87 cells,and the stable expression cell strains were obtained by continuing pressure screening of blasticidin and limiting dilution assay.2. The suppression efficiency of ILK mRNA and protein in BIU-87 stable transfecting cells strains were detected by RT-PCR and Western blot.3. The highest efficienct miRNA interference cell group and non-transfected cell group were acted as research objects. The distribution of cells cycle was analyzed by FCM and the influence on proliferation of BIU-87 cells was investigated by MTT. The proliferation of cells in vivo was analyzed by xenograft tumors experiment in nude mice subcutaneous.Results:1. The cell strains of stable expressing miR-ILK-1,2,3,4 and miR-ILK-neg were obtained.2. ILK mRNA and protein expression were inhibited apparently(P< 0.05 )by miR-ILK-1, 2, 3, 4, and miR-ILK-3 group showed the strongest inhibitory effect as follow: 84.6% and 75.5%, respectively. But in stable expressing miR-ILK-neg cells, ILK mRNA and protein expression were not inhibited(P> 0.05 ).3. Flow cytometry showed that the rate of G0/G1 phase was 67.22±2.29%, and S phase was 9.98±0.85% in transfected group cells;while the rate of G0/G1 and S phase was 49.67±1.57% and 32.68±1.18% in non-transfected group cells, respectively. In transfected group, the number of cells in G0/G1 phase was significantly increased compared with non-transfected group, while that of the S phase was significantly decreased (P<0.05). MTT colorimetric assay showed that the proliferation in transfected group decreased significantly compared with non-transfected group (P<0.05). The xenograft tumors experiment showed that the volume of xenograft tumors in nude mice subcutaneous in transfected group and non-transfected group was 56.67±4.32 mm3 and 289.56±36.49 mm3, while the weight was 0.518±0.03 g, and 1.265±0.02 g, respectively. The proliferation ability of transfected group in vivo decreased significantly compared with non-transfected group (P <0.05).Conclusion:The recombinant plasmid pcDNA^TM6.2-GW/EmGFP -miR-ILK can effectively inhibit the expression of ILK gene, thereby inhibit proliferation of bladder cancer BIU-87 cells.