The Effect Of ShRNA Targeting HIF-1α On Expression Of VEGF And PEDF Of Human Corneal Epithelial Cells Under Hypoxic Condition

Purpose To explore the effect of small hairpin loop RNA(shRNA)targeting hypoxia-induciblefactor-1α (HIF-1α) on the expression of vascular endothelial growth factor (VEGF) andpigment epithelium derived fator (PEDF) in spontaneously immortalized human cornealepithelial cells (S-ihCECs) under hypoxic conditions.We expect to develop new therapy forhypoxia-inducible corneal neovascularization through constructing shRNAs plasmids targetingHIF-1α that transfected into S-ihCECs and then screening the high interference efficiency one.Methods The S-ihCECs were recovered and cultured. The biological characteristics wasobserved under inverted microscope and the expression of cytokeratin12(CK12) byimunofluorescent stain.The proliferation rate of S-ihCECs in various hypoxia conditions wasmeasured by CCK8assay. Qantitative reverse transcription PCR (qRT-PCR) was used to detectthe expression of HIF-1α mRNA in different concentration of CoCl2and imunofluorescent stainwere used to detect the expression of HIF-1α protein in different exposure time toCoCl2.Designed and constructed three kinds of shRNAs targeting sites of HIF-1α mRNA,thenthe shRNAs were transfected into S-ihCECs in vitro.After transfection, the cells were exposuredto1mM CoCl2for6h to induce hypoxia. HIF-1α、VEGF and PEDF mRNA expression weredetected by qRT-PCR. HIF-1α、VEGF protein levels were analysed by Western blotting andPEDF protein level was tested by Enzyme Linked immunosorbent assay (Elisa).Results Most S-ihCECs start growing adherence2h after subculturing. The cells shaped astriangle or fusiform at first,and then spread gradually and became irregular polygon.The cellsdisplayed like pave road stone when they reached80%confluence and expressed CK12. Theproliferation rate of S-ihCECs was increased with the increasing concentration of CoCl2whenthe cells were exposured to CoCl2for6h.The lower concentrations of CoCl2promotedproliferation of S-ihCECs and the higher ones inhibit proliferation when the cells wereexposured to CoCl2for24h.The expression of HIF-1α mRNA was statistically significant whenS-ihCECs was cultured in1mM CoCl2for6h compared to normoxic group.Weak fluorescenceemerged in the nucleus and cytoplasm when S-ihCECs were exposured to CoCl2for1h.Thefluorescence in the nucleus was enhanced gradually for3h and reached its highest for6h.Thefluorescence intensity decreased in the nucleus and hardly any fluorescence in the cytoplasm. After the interference, compared to hypoxia group,the expression of HIF-1α and VEGF mRNAdecreased（70.9±2.8）%,（51.9±15.7）%,respectively. The expression of HIF-1α and VEGFprotein was both down-regulated.The expression of PEDF mRNA increased1.22±0.18fold andthe protein was up-regulated.Conclusions The action time and concentrations of CoCl2have effect on the proliferation ofS-ihCECs,which should be taken into consideration in chemical hypoxia-induced cellsresearch.The expression of HIF-1α mRNA was significant difference when the cells wereexposured to various concentrations. RNAi targeting HIF-1α can effectively silence theexpression of HIF-1α,then reduce the expression of VEGF and up-regulate PEDF,indicatingthat the expression of VEGF and PEDF of S-ihCECs is regulated by HIF-1α.HIF-1is one of thekey factors of hypoxia-inducible corneal neovascularization.The research has screend the highinterference efficiency shRNA plasmids targeting HIF-1α,which provide the probable direactionof therapy for hypoxia-inducible corneal neovascularization.