Construction Of DDEFL1 ShRNA Recombined Adenoviruses And Preliminary Investigations On The Biology Of DDEFL1

DDEFL1 also referred to as UPLC1,ASAP3 and ACAP4,is a new member of the ARF-GAPs family.Like other ARF-GAPs,DDEFL1 contains the BAR,PH,GAP domains and the ANK repeats.Previous studies indicated that DDEFL1 might play pivotal roles in cell migration,vesicle transport,cell skelecton rearrangement and cell proliferation,and might also offer growth advantage to cancer cells under poor nutritional and hypoxic conditions.It has been reported that DDEFL1 was highly up-regulated in several human hepatocellular carcinoma.Conversely,reduced DDEFL1 expression by transfection with anti-sense S-oligo nucleotides inhibited the growth of hepatic SNU423 cancer cells.Several reports also demonstrated the impotant function of DDEFL1 on cell migration.Knockdown of DDEFL1 expression by siRNA significantly attenuated cell migration of HeLa and MDA-MB-231 cells. DDEFL1 has been detected in human liver and lung.We have designed and synthesized three pairs of DDEFL1-shRNAs and then insert them into pSilencer-3.1 H1-neo,a type of eukaryotic shRNA expression vector. By using PCR,we amplified the RNAi expressing cassette which contained H1 promoter and DDEFL-shRNA.Further,the cassettes were inserted into pAd-Track vector and then pAd-DDEFL1-shRNA was attained by homologous recombination. Finally,recombined adenoviruses were packaged in AD293 cells and we got three recombined adenoviral vectors expressing DDEFL1-shRNA(Ad1,Ad2 and Ad3) and a negative control containing scrambled sequence(Ad4).To further investigate the roles of DDEFL1 in cell migration,proliferation and cell cycles,we knocked down DDEFL1 level of HepG2 and H1299 cells by adenoviral mediated DDEFL1 RNAi. RT-PCR and Western Blot were applied to determine DDEFL1 mRNA and protein level,respectively.Then we used wound healing assay to measure cell migration activity.MTT and flow cytometry were used to determine cell proliferation,cell cycles and apoptosis.Our RT-PCR and Western Blot showed that DDEFL1 was significantly expressed in both HepG2 and H1299 cell lines.Transduction with recombined adenoviruses containing DDEFL1-shRNA could reduce mRNA level and protein level of DDEFL1 in HepG2 and H1299 respectively.Among three shRNA recombinant viruses,Ad3 has the best effect on reducing DDEFL1 expression and suppressed the DDEFL1 mRNA level by 67%(P＜0.005) in HepG2,and suppressed the DDEFL1 protein level by 61%(P＜0.05) and 75%(P＜0.05) in HepG2 and H1299 respectively while Ad4 has no evidence effect on DDEFL1 expression.Reduction of DDEFL1 expression had no effect on cell proliferation and cell-cycle of H1299,but the expression of DDEFL1 could provide a growth advantage to cancer cells in low glucose nutritional. Furthermore we found that adenovirus mediated DDEFL1 RNAi inhibited migration of H1299 obviously,H1299 with reduced DDEFL1 migrate into the wound is much slow than Ad4 traded or control cells,and similar results were obtained in HeLa and HepG2 cells.