Effect of down-regulation of integrin β1 on the migrative and adhesive capacity of non-small lung cancer cells / 肿瘤

ABSTRACT

Objective:To construct RNA interference plasmid specific for integrin β1 gene, and to explore the effects of inhibition of integrin β1 protein expression on the biological behavior of non-small cell lung carcinoma(NSCLC) cells. Methods:Genomic sequences of integrin β1 gene was retrieved from GenBank, and cDNA encoding small hairpin RNA(shRNA) for integrin β1 was designed and named as 17-2. A non-specific sequence was designed as negative control and named as N. The cDNA was synthesized and inserted into plasmids pUC19. Recombinant plasmids were then transformed into competent E.coli. The positive clones were selected and recombinant plasmids were extracted.The two shRNA vectors were transfected into NSCLC cell line PC-9/AB2 mediated by lipofectAMINE 2000. The stably transfec-ted cell clones were selected by G418 screening. Fluorescence microscope, real-time fluorescent quantitative (RFQ)-PCR, and Western blotting were performed to examine integrin β1 mRNA and protein expressions.Cell scratch test and adhesion test were used to detect the influences of silencing integrin β1 on cell migration and adhesion capacity. Results:The stably transfected 17.2 cell clones and N cell clones were screened by G418. The green fluorescence-staining cells were observed in full-field under fluorescence microscope. The level of integrin β1 was significantly down-regulated in 17-2 group compared with N group and primary PC-9/AB2 cells.Scratch test and adhesion test showed that the migration and adhesion capacity of PC-9/AB2 cells was significantly reduced after silencing integrin β1. Conclusion:This study successfully constructed integrin β1 specific shRNA expression vector. The expression of integrin β1 was significantly silenced in NSCLC cells transfected with that vecton. Silencing integrin β1 can significantly reduce the migration and adhesion capacity of NSCLC cells.