The Role And Mechanism Of Gene CREB1on Oxygen Induced Retinal Neovascularization In Mice

Part One CREB1expression in retinas of mice with oxygen induced retinopathyPurpose This study aimed to explore the relation between the expression of CREB1and retinal neovascularization(RNV) in OIR mouse model.Methods Postnatal day7(P7) mice (n=134) were randomly assigned to two groups:control group (n=67) and OIR (oxygen induced retinopathy, OIR) group (n=67). Mice from OIR group were exposed to (75±2)%oxygen for5days and then to room air for additional9days. The mice from control group were raised in the normal environment for21days. The P17mice from two groups were sacrificed, retinal sections for HE staining and flat mounts after cardiac perfusion with FITC-dextran were used to detect retinal neovascularization. Immunofluorescence of retinal frozen sections showed the expression and location of P-CREB1protein. Real-Time PCR and Western blot were used to detect the expression of CREB1in retinas. Independent sample t test was used to analyze the data from different groups at the same time.Two-Way ANOVA was used to analyze the data from two groups at different time points.Bonferroni post-test was used for comparing the data between two time points.Results The numbers of the cellular nuclei of vascular endothelium breaking through the retinal internal limiting membrane were significantly higher in OIR group than in control group at P17, a statistically significant difference (t=11.31, p<0.05). Areas of retinal new blood vessel and avascular zones were (30.61±3.12)%and (21.40±2.72)%respectively in OIR mice at P17. P-CREB1protein was more strongly expressed in INL and GCL of retinas from the OIR group than that of the contral group.The results of real-time PCR and Western-blotting showed that the relative expression level of mRNA and protein of CREB1gradually increased from P7to P17, followed by decreased expression at P21in control group. But the values were significantly higher in OIR group than the control group in various aged mice except for P7mice (P<0.05).Conclusions These results indicate a space-time corresponding relation between the expression of CREB1and retinal neovascularization. The overexpression of CREB1 could be involved in the process of retinal neovascularization in OIR model. Part Two Construction, packaging, identification of RNA small interference based on CREB1lentivirus vectorObjective:This study was to design, construct and package RNAi lentiviral expression vector aming the mouse CREB1gene, and to provide experimental tool and theoretical proof for the further experiment.Methods:1. Plasmid, pLenR-GPH Vector, was used to construct the recombinational expression vector of CREB1siRNA.2. Two CREB1siRNA were designed to be consisted of two specific siRNA target sequences by the mRNA nucleotide sequence for the mouse CREB1gene in the NCBI. Double-stranded DNA oligo with the interfering sequence was synthetized and linked to the enzyme digested vector directly.3. Linked plasmids were transferred into competent cells. The positive clones were acquired and identified by PCR. The plasmids were extracted and identified by sequencing after positive clones were amplificated.4. The positive clones were identified as the succesfully constructed lentiviral vector of CREB1siRNA by the sequencing comparison.5. The titer of recombinational CREB1siRNA lentivirus was determined by Flow cytometry.Result:The gene CREB1siRNA lentiviral vector carrying EGFP was constructed and packed successfully.The titer of recombinational lentiviral vector was≥1.5×109TU/ml.Conclusion:Lentiviral vector carrying CREB1gene siRNA with EGFP was constructed and packed successfully. The titer of recombinational CREB1siRNA lentivirus was high and meeted the animal experimental needs. Recombinational CREB1siRNA lentivirus provided a basic experimental tool for further investigation of gene regulation by RNA interference. Part Three The inhibition and mechanism of lentivirus-mediated siRNA targeting CREB1on retinal neovascularization in mice with oxygen induced retinopathyObjective To investigate the inhibitory effect and mechanism of lentivirus-mediated siRNA targeting CREB1on retinal neovascularization in mice with oxygen induced retinopathy by intravitreal injection.Methods One hundred and forty (5-day-old) C57BL/6J mice were randomly divided into4groups including the normal group, the OIR model group, the empty vector group and the CREB1therapy group with35mice in each group. Mice in the normal group were kept in normal room air, while in the other three groups retinal neovascularization was induced by establishing the oxygen induced retinopathy model according to the method we previously mentioned. The mice in the OIR model group were not treated. The mice in the empty vector group received intravitreal injection of lentivirus without CREB1siRNA (CREB1-NC-siRNA,1μl), and the CREB1therapy group received intravitreal injection of lentivirus with CREB1siRNA (CREB1-siRNA,1μl) on P5. The transfection of lentiviral vector was examined under fluorescence microscope on P12. The expression of phosphorylated-CREB1(P-CREB1) protein was detected for evaluating the interference efficiency by immunofluorescence on P17. The proliferative neovascular response was quantified by counting the vascular cell nuclei extending breaking through the internal limiting membrane (ILM) and fluorescent angiography. The areas of RNV and non-perfusion region were calculated. The expression of CREB1, P-CREB1and vascular endothelial growth factor (VEGF)-A levels, Akt and phosphoinositide3-kinases (PI3K) in retinas were measured by real-time quantitative polymerase chain reaction (RT-PCR) and Western blot. Data was analyzed with a one-way ANOVA.Results Expression of GFP was detected on the7th day after intravitreal injection of lentiviral vector. GFP was expressed highly in the CREB1therapy group and the empty vector group. There was no GFP in the normal group and the OIR group. The fluorescence of P-CREB1was more strongly expressed in inner nuclear layer (INL) and ganglion cell layer (GCL) of retinas from the OIR model group and the empty vector group than that from the CREB1therapy group,and it was faint in the retinas from the normal group on P17.The numbers of VEC nuclei breaking retinal ILM were significantly different among the OIR model group, the CREB1therapy group and the empty vector group (F=50.48,p<0.01). Fluorescent angiography showed that the distribution of retinal vessels was uniform and there was no vessel obliteration, neovascularization and avascular zone in the normal group, but abnormal vessels could be observed in other three groups. The area of retinal neovascularization and avascular zone in the CREB1therapy group was significantly reduced compared with the OIR group and the empty vector group (F=110.09, P<0.01; F=67.22, P<0.01). There was no significant difference in the areas of retinal neovascularization and avascular zone between the OIR group and the empty vector group (p=0.917;p=0.877). The mRNA expression of CREB1and protein expression of P-CREB1, the mRNA and protein expression of VEGF-A, Akt, PI3K in the retina were increased significantly in the OIR group and the empty vector group compared with the normal group, while decreased significantly in the CREB1therapy group compared with the OIR group and the empty vector group. The difference of mRNA expression of CREB1, VEGF-A, Akt, PI3K in the retina among4groups were significant (F=6.087,5.464,6.191,8.627;<0.05). The protein expression of P-CREB1, VEGF-A, Akt, PI3K in the retina among4groups were significant(F=162.944,13.861,19.710,22.827;<0.05).Conclusions RNV in the mice with OIR is significantly inhibited by intravitreal injection of lentivirus-mediated CREB1siRNA.This role was related to the down-regulation of PI3K/Akt and VEGF-A.