The Role Of Hox Genes In High Glucose Condition Inducing Differentiation And Proliferation Of Cultured Human Retinal Pigment Epithelial Cells

BACKGROUNDAs the incidence of diabetes worldwide continuing to rise, diabetic retinopathy (DR) has become one of the most common cause of blindness after birth。over 90% of people with diabetes can cause retinopathy, of which about 60% of type 1 diabetes,20% in patients with type 2 diabetes will eventually progress to proliferative diabetic retinopathy (PDR),and PDR in our country has already become the key point to treat and prevent blindness currently as well as in future.However,given the current treatment on PDR, laser photocoagulation and vitrectomy can injure local tissues and their long-term efficacy is not available, it is quite necessary to look for new approaches for treatment based on its pathogenesis.PDR is a kind of cell proliferative diseases with neovascularazation as the main pathological characteristics.It is related to cell activation, proliferation and differentiation control disorders, and new vessels are also the results of abnormal cell proliferation. Type I Hox genes(Hox genes),a vital kind of growth-related genes, contribute to determine the orientation of cell differentiation and proliferation. Dysfunction of Hox genes or their transcriptional regulation protein will directly affect cell proliferation and differentiation progress. Studies have shown that Hox genes have been involved in the progress of angiogenesis, especially playing an important role in the anti-angiogenic targeted therapy of tumor. They have also been involved in body and organ morphogenesis in embryocic development, etc. And they are responsible for the regulation of the hematopoietic proliferation and differentiation as race-specific and stage-specific manner. There are hitherto rare studies about the relationship between Hox genes and diabetes.And the relationship between PDR and Hox genes has not yet been reported. Studies show that ARPE-19 cells grown in hyperglycemic conditions may display distinct cell differentiation and cell functions, including high glucose causes the changes of RPE cells to secrete cytokines such as vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF) expression and the aspect of promoting RPE cells proliferation,etc. Thus we assume Hox genes is involved in the progress. PDR is a kind of disease associated with neovascularization. VEGF and PEDF are major blood vessels stimulating factor and inhibiting factor respectively, the balance between which is crucial to angiogenesis. Lots of studies have proved that Hox genes are involved in angiogenesis, parts of which have reported they may regulate the expression of VEGF or PEDF. Theremore, we assume Hox genes contribute to angiogenesis by regulating the expression of VEGF or PEDF. In order to validate the hypothesis, we design the experiments to investigate the pathogenesis of PDR, and then we may find an effective treatment and prevention for it.OBJECTIVE1) To investigate the effect of different concentrations of glucose on proliferation and differentiation of ARPE-19 cells, as well as the expression of VEGF and PEDF.2) To investigate the expression of Hox genes in ARPE-19 cells cultured in different concentrations of glucose cultured media in order to explore the relationship between Hox genes and PDR. To choose the hox gene, the expressional difference of which is the most obvious, to the next experiment.3) To investigate the effect of the purpose gene on proliferation and differatiation of ARPE-19 cells as well as the expression of VEGF and PEDF.To explore its possible relative mechanisms。METHODS1) ARPE-19 cells cultured in 18mM and 5.5mM DMEM respectively. Comparing the number of proliferation and cell morphology per day (1-8 days) between the two groups. ARPE-19 cells were divided into groups according to the concentration of glucose in the culture media:1.the control group(high glucose group(HG,18mM)); 2.the experiment groups(normal glucose group(NG,5.5mM)):NG1(cultured 7 days cell)group; NG2(cells cultured for 14 days) group; NG3(cells cultured for 21 days) group.RT-PCR and western blot were used to detect the expression of VEGF and PEDF in all groups.2) HG and NG were experiment group and control group, respectively. Application of Hox gene family-specific primers to detect the mRNA expression levels of 39 genes in the two groups by RT-PCR and image analysis. The level of the gene expression was expressed as ratio expression rate (RER) of Hox gene/GAPDH to computer image analysis.The difference between the two groups was analyzed by Statistical analysis.The expression level of HoxB7, the expressional difference of which is the most available, was measured by western blot.3) Cells were divided into five groups:ARPE-HOXB71, ARPE-HOXB72, ARPE-HOXB73, ARPE-Neg and non-transfected control group. Three HoxB7 specific small interfering RNA (small interfering RNA, siRNA) and a negative control (negtive-siRNA) were designed according to its mRNA sequence, then cloned into pRNAT plasmid vector. The recombinant plasmids were transfected into ARPE-19 cells, respectively. The transfection efficiency was detected with fluorescence microscopy.RT-PCR and western blot were used to confirm the effect of HoxB7 siRNA and to detect the expression of VEGF and PEDF. Using MTT investigate d the effect of HoxB7 on cell proliferation in all groups. To compare cell morphology between every group.RESULTS1) Cells grown in high glucose had an elongated or spindle shape. Cells grown in physiological glucose concentrations had a rounded or cobblestone shape. A significant difference in cell number between the two groups was noted by the fourth day of the experiment (p<0.05).Cells grown in high glucose increased in viable cell number from 5.1±0.3E+04/ml on day 1 to 31.2±2.1E+04/ml on day 8. The cells in 5.5 mM glucose increased from 4.5±0.4E+04/ml per well on day 1 to 14.1±1.2E+04/ml per well by day 8. The expression of PEDF-mRNA in NG was higher than HG, and NG1 comparing with HG (P<0.05), NG2 and NG3 comparing with HG (P<0.01); the expression of PEDF ptotein in NG was higher than HG (P<0.01), and the expression level increased with the time (P<0.01); the expression of VEGF in NG was lower than HG, and the expression level decreased with the time (P<0.01).2) There are 12 hox genes(HoxA5,HoxA6,HoxA13,HoxB3,HoxB5,HoxB7,HoxB13,HoxC6,HoxC13,HoxD1,HoxD3,HoxD10) expressed in two groups. The expressional difference of(HoxA5,HoxA13,HoxC13,HoxD1,HoxD3) between two groups were not significant(P>0.05). The expression of HoxA6,HoxB3,HoxC6,HoxD10 in HG were down-regulated comparing with NG(P<0.05). The expression of HoxB5,HoxB7,HoxB 13 in HG were up-regulated comparing with NG(P<0.05).The expressional difference of HoxB7 is most obvious.3) pRNAT-HoxB71 knocked down expression of HoxB7 specifically and effectively. Comparing with negative control group and blank group, the level of HoxB7 mRNA and protein in ARPE-19 cells transfected stably with pRNAT-HoxB71 were inhibited (50.15±2.31)% and (55.41±2.99)%. The level of VEGF mRNA and protein in ARPE-19 cells transfected stably with pRNAT-HoxB71 were lower than negative control group and blank group (P<0.01). The level of PEDF mRNA and protein in ARPE-19 cells transfected stably with pRNAT-HoxB71 were higher than negative control group and blank group (P<0.01). Comparing with negative control group and blank group, knocking down HoxB7 expression by siRNA inhibited proliferation of ARPE-19 cell. The inhibition rations were (16.18±2.53)% and (25.02±5.02)% respectively after the transfection time of 48h and 72h. There was no difference in cell morphology comparing with negative control group and blank group after knocking down HoxB7 expression by siRNA.CONCLUSION1) High glucose can stimulate the proliferation and differentiation of hRPE cell. To control the blood glucose level can up-regulate PEDF and suppress VEGF on the level of transcription and translation in hRPE cells.2) There are 12 hox genes (HoxA5,HoxA6,HoxA13,HoxB3,HoxB5,HoxB7,HoxB13,HoxC6,HoxC13,HoxD1,HoxD3,HoxD10) that may relate to the proliferation and differentiation progress in hRPE cells. HoxA6,HoxB3,HoxB5,HoxB7,HoxB13,HoxC6 and HoxD10 may have relationship with PDR. HoxB7 may be one of the members most closely related to PDR.3) The effect that high glucose can stimulate the proliferation and differentiation of hRPE cell may be achieved by up-regulating the expression of HoxB7. HoxB7 may have relationship with PDR by up-regulating PEDF and suppressing VEGF in hRPE cells.