| PrefaceDiabetic retinopathy(DR)is one of the common microvascular complications of diabetes mellitus,which is the main cause of blindness in diabetic patients.With the rising incidence of diabetes,DR has become the main cause of blindness and visual impainnent in the working age population.DR currently affects almost 100 million people worldwide and is becoming an ever-increasing health burden,with estimates between 1990 and 2010 showing that DR-related visual impairment and blindness increased,respectively.Therefore,a comprehensive understanding of DR is urgently needed and.will enable the development of novel diagnostic and effective therapeutic strategies to prevent DR progression and improve patients’ prognosis.Given this substantial clinical problem,many researchers are focused on resolving molecular mechanisms in DR pathogenesis to identify potential new therapeutic targets.Part I expression of MiR-200a and PDLIM1 in DRObjectiveTo verify that MiR-200a and PDLIM1 are involved in the course of DR.MethodsFrom January 2019 to January 2020,30 diabetic patients,patients with retinopathy,healthy people and patients with idiopathic macular anterior membrane were collected,which were DM group,DR group and control group and IERM group respectively.The expression of MiR-200a and PDLIM1,inflammatory factors and apoptosis factors Bax,BCL2 and Caspase-3 in the serum of the patients were detected.The data between the two groups were compared by independent sample t-test,and the data of more than two groups were compared by one-way ANOVA.1ResultsThe MiR-200a of DR group was lower than that of IERM group,and the PDLIM1 expression of DR group was higher than that of IERM group(P<0.05).The luciferase activity of MiR-200a+PDLIM1 co transfection group was significantly lower than that of MiR-nc+PDLIM1 co transfection group(P<0.05).The levels of MiR-200a and PDLIM1 in DM group and DR group were significantly lower than those in control group(P<0.05).MiR-200a was negatively correlated with PDLIM1 expression.The expression of IL-1β,TNF-α,IL-6 and ICAM-1 in DM group and DR group were significantly higher than that in control group,but there was no significant difference between DM group and DR group.The expression of Bax and caspase-1 in DM,DR group was higher than that in control group,DR group was significantly higher than DM group(P<0.05),the expression of Bcl-2 in DM,DR group was significantly lower than that in control group(P<0.05),DR group was significantly lower than DM group。Conclusion1.MiR-200a and PDLIM1 are abnormal in DR,and they participate in the development of DR,they are negatively correlated,which is worthy of further study.2.The high expression of inflammatory factors in DR patients is one of the causes of DR.3.All kinds of retinal cells in DR patients are in a high level of apoptosis.Part II MiR-200a relieves hyperglycemic induced inflammation and permeability of HRMECS through PDLIM1ObjectiveTo investigate the expression of MiR-200a and PDLIM1 in human retinal microvascular endothelial cells(HRMECS)in high glucose environment,and the effects of MiR-200a and PDLIM1 on cell activity,apoptosis,migration and oxidative stress inflammatory factors.MethodsHRMECS were used as experimental cells to detect the expression of MiR200a and PDLIM1 in the cell model of high glucose culture.Four groups of control,Hg,Hg+MiR-nc and Hg+MiR-200a were used to construct wild-type and mutant PDLIM1 plasmids.The target gene of MiR-200a was analyzed by double Luciferase Report,Control group,ng+MiR-nc group,ng+MiR-200a group,high glucose group(Hg),Hg+MiR-nc group,Hg+MiR-200a group were set up.In each group,endothelial cells were cultured in different concentrations of glucose(0mnol/L,0mnol/L,0mnol/L,35mnol/L,35mnol/L,35mnol/L)for 24 hours to construct high glucose cell model.PDLIM1,ng+MiR-200a,MiR-nc plasmids were designed and transfected.MTT was used to detect cell survival rate,flow cytometry was used to detect apoptosis rate,and Bax,BCL2,caspase were detected by Western blot The expression of IL-1 β,TNF-α,IL-6 and ICAM1 were detected by ELISA.T test was used to compare the data differences between the two groups,and single factor analysis of variance was used to compare the data differences between multiple groups.ResultsMiR-200a and PDLIM1 were expressed in the high glucose culture cell model,and with the increase of the time of high glucose treatment,the expression of MiR-200a decreased and PDLIM1 increased.Compared with the control group,the activity of cells treated with PDLIM1 had no significant change,apoptosis rate,migration rate,ROS and inflammatory factor expression increased(P<0.05);the activity of cells treated with MiR-200a increased,apoptosis rate,migration rate decreased,ROS and inflammatory factor expression decreased(P<0.05).Conclusion1.The expression of MiR-200a decreased and PDLIM1 increased in2.HRMECS in high glucose environment.3.PDLIM1 is the target gene of MiR-200a.MiR-200a regulates PDLIM1 and affects DR in the DR process.MiR-200a can inhibit apoptosis and migration of HRMECS cells in high glucose environment,and improve cell viability,and inhibit the secretion of ROS and inflammatory factors IL-1β、IL-6、TNF-α、ICAM-1 in HRMECS cells in high glucose environment.Part III MiR-200a affects DR process by downregulating PDLIM1ObjectiveTo investigate the expression of MiR-200a and PDLIM1 in the retina of diabetic mice and the effects of MiR-200a and PDLIM1 on the thickness and permeability of retina,typical inflammatory factors such as IL-1β,TNF-α,IL-6 and ICAM1.MethodsC57BL/6J male mice of 7-8 weeks old were injected with STZ intraperitoneally to construct the model of type 2 diabetic mice.Blood glucose and body weight were detected.MiR-200a and PDLIM1 contents of DR mice were detected by RT-PCR at 2 and 4 months.The content of PDLIM1 in the retina of DR mice was detected by immunohistochemistry.Control group,high glucose group,DM+PDLIM1 group,DM+MiR200a group,DM+PDLIM1+MiR-200a group,DM+PDLIM1+MiRnc group,respectively,were injected with 1μl plasmids of MiR-200a MiR-nc and PDLIM1 in the neck,OCT was used to detect the retinal thickness,Evans blue was used to detect the retinal vascular permeability,and ELISA was used to detect the inflammatory factors in the vitreous.T test was used to compare the data differences between the two groups,and single factor analysis of variance was used to compare the data differences between multiple groups.ResultsThe expression levels of MiR-200a and PDLIM1 were detected at different time points in diabetic mice retinopathy model.The expression of MiR-200a decreased at 2 and 4 months after STZ injection,while the expression of PDLIM1 increased significantly at 2 and 4 months(P<0.05).PDLIM1 distribution in mouse retina increased with time,less in control group and more significantly in DM group.The retinal thickness,permeability and expression of inflammatory factors increased in DM+PDLIM1+MiR-200a group compared with DM+PDLIM1 group The retinal thickness,permeability and expression of inflammatory factors were significantly decreased(P<0.05).PDLIM1 treatment could significantly increase the retinal thickness,and MiR-200a injection could improve the effect.Conclusion1.With the increase of time,MiR-200a expression decreased in the retina of diabetic rats,and PDLIM1 expression increased in DR.2.PDLIM1 can increase the retinal thickness and permeability of diabetic mice,and promote the development of DR course.MiR-200a can inhibit the effect of PDLIM1 on the retina of diabetic mice,reduce the retinal thickness and permeability,and delay the development of DR course.3.PDLIM1 can increase the content of inflammatory factors such as IL-1β,IL-6,TNF-α and ICAM-1 in the retina of diabetic mice,and promote the development of DR.MiR-200a can inhibit the effect of PDLIM1 on inflammatory factors in the retina of diabetic mice,reduce the expression of inflammatory factors such as IL-1β,IL-6,TNF-α and ICAM-1,and delay the development of DR.Part IV PDLIM1 participates in DR progress by regulating Wnt signaling pathwayObjectiveIn vitro cell experiments were carried out to verify the effect of PDLIM1 on the key proteins and downstream genes of Wnt signaling pathway,and its role in the course of DR and inflammation.MethodsHRMECS were used as experimental cells,and four groups were set up:control group,PDLIM1 overexpression group,PDLIM1 knockdown group and negative virus group.The expression of E-cadherin,β-Catenin,cyclin D1,c-myc and COX-2 in Wnt signaling pathway were detected in each group after cultured in EGM for 24 hours.Control group,ng+MiR-nc,ng+Si β-Catenin group,high glucose group(Hg),Hg+MiR-nc,Hg+Si β-Catenin group were set up.Each group was transformed into endothelial cell culture medium with different concentrations of glucose(0mnol/L,0mnol/L,0mnol/L,35mnol/L,35mnol/L,35mnol/L)for 24 hours to construct high glucose cell model,design and transfect Si β-Catenin and MiR-nc,PDLIM1 plasmid was used to detect cell survival rate by MTT,apoptosis rate by flow cytometry,ROS content,and the expression of inflammatory factors IL-1β,TNF-α,IL-6 and ICAM1 in vitreous by ELISA.The data between the two groups were compared by independent sample t-test,and the data of more than two groups were compared by one-way ANOVA.ResultsIn PDLIM1 group,the expression of E-cadherin,β-Catenin protein and downstream genes cyclin D1,c-myc and COX-2 increased,while in Si PDLIM1 group,the expression of E-cadherin,β-Catenin protein and downstream genes cyclin D1,c-myc and COX-2 decreased(P<0.05).Inhibition of β-Catenin protein in Wnt pathway can increase the activity of HRMECS cells in high glucose environment,reduce the apoptosis rate and the expression of ROS and inflammatory factors,and inhibit the effect of PDLIM1 on cells.Conclusion1.PDLIM1 affects the use of Wnt signaling pathway in DR process,which can reduce the expression of genes downstream of Wnt signaling pathway.2.Inhibition of Wnt signaling pathway can inhibit the effect ofPDLIMlon HRMECS cell activity and inflammatory factors. |
