MET Inhibits Proliferation,Migration And Angiogenesis Of RF/6A Cells Induced By High Glucose And Related Mechanisms

Objective: To investigate the effects of metformin(MET)on the proliferation,migration and angiogenesis of choroid-retinal endothelial cells(RF/6A)induced by high glucose in rhesus monkeys,and to explore its mechanism preliminarily.Methods: The RF/6A cells were divided into five groups: normal control group(NC group),high glucose group(HG group),high glucose plus different concentrations of metformin hydrochloride group(HG + 15,30,45 mmol/L MET group).Cell proliferation was detected by CCK-8 method.The RF/6A cells were divided into four groups: normal control group(NC group)and normal control group with different concentrations of metformin hydrochloride(NC + 15,30,45 mmol/L MET group).Cytotoxicity was detected by CCK-8method.The RF/6A cells were divided into three groups: normal control group(NC group),high glucose group(HG group)and high glucose plus 15 mmol/L metformin hydrochloride group(HG + 15 mmol/L MET group).Cell migration was detected by cell scratch method and Transwell method,angiogenesis was detected by Matrigengel method,and the expression of m RNA and protein of YAP and TEAD1,the core components of the Hippo pathway,were detected by RT-PCR and Western blot methods.Results: The results of CCK-8 method showed that the proliferation activity of RF/6A cells in HG group was significantly higher than that in NC group(P < 0.001),and the proliferation activity of RF/6A cells in HG + 15,30 and 45 mmol/L MET groups was lower than that in HG group(all P < 0.001).There was no statistical significance between NC group and NC + 15 mmol/L MET group(P = 0.2273,P > 0.05),indicating that 15 mmol/L metformin hydrochloride was non-toxic to RF/6A cells,so 15 mmol/L was selected for follow-up experiment.The results of cell scratch method and Transwell method showed that the migration rate and number of RF/6A cells in HG group were significantly higher than those in NC group(both P < 0.01),and the migration rate and number of RF/6A cells in HG + 15 mmol/L MET group were significantly lower than those in HG group(both P < 0.01).The results of Matrigengel method showed that the relative total tube length of RF/6A cells in HG group was significantly higher than that in NC group(P < 0.05),and the relative total tube length of RF/6A cells in HG+ 15 mmol/L MET group was significantly lower than that in HG group(P< 0.01).The results of RT-PCR method showed that the relative expression of YAP m RNA in RF/6A cells was 1.000 ± 0.058 in NC group and 1.169 ± 0.018 in HG group.There was significant difference between the two groups(P < 0.05).And the relative expression of YAP m RNA in HG + 15 mmol/L MET group(1.068 ± 0.018)was significantly lower than that in HG group(P < 0.05).The relative expression of TEAD1 m RNA of RF/6A cells in NC group was 1.000 ±0.014,and the relative expression of TEAD1 m RNA in HG group was1.097 ± 0.016.There was significant difference between the two groups(P < 0.01).And the relative expression of TEAD1 m RNA in HG + 15mmol/L MET group(0.985 ± 0.006)was significantly lower than that in HG group(P < 0.01).The results of Western blot method showed that the relative expression of YAP protein in RF/6A cells in HG group(1.374 ± 0.050)was significantly higher than that in NC group(1.000 ±0.044)(P < 0.01),while the relative expression of YAP protein in HG +15 mmol/L MET group(1.190 ± 0.044)was significantly lower than that in HG group(P < 0.05).The relative expression of TEAD1 protein in RF/6A cells in HG group(1.154 ± 0.047)was significantly higher than that in NC group(1.000 ± 0.0287)(P < 0.05),while the relative expression of TEAD1 protein in HG + 15 mmol/L MET group(0.769 ±0.055)was significantly lower than that in HG group(P < 0.01).Conclusion: Metformin can inhibit the proliferation,migration and angiogenesis of RF/6A cells induced by high glucose,and down-regulate the m RNA and protein expression of YAP and TEAD1.It is speculated that its mechanism is related to Hippo signal pathway.