Construction of lentiviral vector of RNA inter-ference of MMP-9 gene and its inhibitive role on the invasion of laryngeal cancer cell / 中国耳鼻咽喉头颈外科

ABSTRACT

OBJECTIVE To construct a lentiviral vector of RNA interference (RNAi) of MMP-9 gene and observe its inhibitive role on the invasion of laryngeal cancer cells.METHODS The effective sequence of siRNA targeting MMP-9 gene was confirmed.Both sense and antisense Oligo DNA of the targeting sequence was designed,synthesized and cloned into the pLVTHM vector,which contained H1 promoter and green fluorescent protein (GFP).The resulting lentiviral vector containing MMP-9 shRNA was called LV2 shMMP-9,and it was confirmed by PCR and sequencing.After that,MMP-9 shRNA was transfected into Hep-2 cells and western blot was used to test the expression of MMP-9.At last,Boyden Chamber was used to observe the invasion of the Hep-2 cells. RESULTS PCR and DNA sequencing demonstrated that the lentivirus RNAi vector of MMP-9 (LV2shMMP-9) producing MMP-9 shRNA was constructed successfully. The titer of concentrated virus was 8?1010TU /L. Western blot showed that the expression of MMP-9 was negative in the MMP-9 siRNA transfected Hep-2 cells. And Boyden Chamber showed the invasive capability of Hep-2 cells transfected MMP-9 siRNA were obviously decreased.CONCLUSION The lentivirus RNAi vector of MMP-9 was constructed successfully,and MMP-9 silence can inhibit invasion of laryngeal cancer in vitro.