title: Annexin A2 silencing enhances apoptosis of human umbilical vein endothelial cells in vitro.
abstract: OBJECTIVE
  To study the effects of inhibited Annexin A2 (ANXA2) on human umbilical vein endothelial cells (HUVECs) in vitro.
  
  
  METHODS
  Short hairpin RNA (shRNA) targeting ANXA2 was designed and cloned into double marked lentivirial vector GV248 for RNAi to generate the recombinant expression plasmids, which were stably transfected into HUVECs. The protein and mRNA expression levels of ANXA2 were analyzed by western blotting and real-time polymerase chain reaction, respectively. Cell proliferation (cell counting kit-8 assay), apoptosis (flow cytometry analysis), the expression (western blotting) and the activity of caspases (enzyme-linked immunosorbent assay) were used to assess the effects of silencing ANXA2 on HUVECs in vitro.
  
  
  RESULTS
  The plasmids to express ANXA2-specific shRNA were constructed and were infected into HUVEC resulting in the stably transfected experimental (ANXA2-shRNA), control (control-shRNA) and mock (no plasmid) cell lines, which were verified with western blot and real-time PCR. HUVEC/ANXA2-shRNA showed an inhibition rate 91.89% of ANXA2 expression compared to the mock HUVEC. ANXA2 silencing cell strain obviously presented a lower cell proliferation activity compared to the control and mock HUVECs, with an inhibition rate 82.35% on day 7 in vitro. FACS analysis indicated that the HUVEC/ANXA2-shRNA cells undergoing apoptosis increased by 102.61% compared to the mock HUVECs (P < 0.01). Moreover, the activity levels of caspase-3, caspase-8 and caspase-9 in HUVEC/ANXA2-shRNA cells were increased and the activated cleaved caspase-3, cleaved caspase-8 and cleaved caspase-9 were upregulated evidently compared with that of the control and mock HUVECs by 56.29%, 89.59% and 144.58% (P < 0.01).
  
  
  CONCLUSIONS
  shRNA-mediated silencing of ANXA2 could not only be able to suppress HUVECs proliferation but to upregulate the enzyme activity of caspases, which bring to an increase of cell apoptosis. This work suggested that ANXA2 may represent a useful target of future molecular therapies.
authors.affiliation: Department of Ophthalmology, Affiliated Changhai Hospital of Second Military Medical University, Shanghai, 200433, China.
authors.forename: Shu-Le
authors.initials: SL
authors.lastname: Jiang
authors.affiliation: Department of Ophthalmology, Affiliated Changhai Hospital of Second Military Medical University, Shanghai, 200433, China.
authors.forename: Dong-Yan
authors.initials: DY
authors.lastname: Pan
authors.affiliation: Department of Ophthalmology, Affiliated Changhai Hospital of Second Military Medical University, Shanghai, 200433, China.
authors.forename: Chao
authors.initials: C
authors.lastname: Gu
authors.affiliation: Department of Ophthalmology, Affiliated Changhai Hospital of Second Military Medical University, Shanghai, 200433, China.
authors.forename: Hai-Feng
authors.initials: HF
authors.lastname: Qin
authors.affiliation: Department of Ophthalmology, Affiliated Changhai Hospital of Second Military Medical University, Shanghai, 200433, China. Electronic address: zhaosh2000@sina.com.
authors.forename: Shi-Hong
authors.initials: SH
authors.lastname: Zhao
keywords: Annexin A2; Cell apoptosis; Gene silence; RNA interference; Retinal neovascularization