ABSTRACT: To construct a lentivirus vector of RNA interference (RNAi) of EGFL7 gene and observe its inhibitive role on the invasion of laryngeal cancer cell.
The effective sequence of siRNA targeting EGFL7 gene was confirmed. Both sense and antisence Oligo DNA of the targeting sequence was designed, synthesized and cloned into the pLV vector,which contained H1 promotor and green fluorescent protein (GFP). The resulting lentivirus vector containing EGFL7 shRNA was called LV-sh EGFL7,and it is confirmed by PCR and sequencing. After that, EGFL7 shRNA was transfected into Hep-2 cells and Western blot was used to test the expression of EGFL7. At last, boyden chamber was used to observe the invasion of the Hep-2 cells. Colony formation assay using a EGFL7 gene silencing on the Hep-2 cell colony forming ability.
PCR and DNA sequencing demonstrated that the lentivirus RNAi vector of EGFL7 (LV-sh EGFL7) producing EGFL7 shRNA was constructed successfully. The titer of concentrated virus was 5 x 10(8) TU/L. Western blot showed that the expression of EGFL7 was negative in the EGFL7 siRNA Hep-2 cells. And boyden chamber showed the invasive capability of Hep-2 cells transfected EGFL7 siRNA were obviously decreased. EGFL7 gene silencing of cell colony formation rate of cloned Hep-2 cells and compared with empty vector cells, cell cloning and colony formation was significantly reduced.
The lentivirus RNAi vector of EGFL7 was constructed successfully. And EGFL7 silence can inhibit invasion of laryngeal cancer in vitro. After silence EGFL7, Hep-2 cell colony formation was significantly lower, that is, gene expression can be down EGFL7 some extent laryngeal cancer cells inhibited anchorage independent growth capacity.
Lin chuang er bi yan hou ke za zhi = Journal of clinical otorhinolaryngology 12/2011; 25(24):1135-8, 1141.