| ?Objective?To observe the expression of microRNA(miRNA)-1470(miR-1470)in the plasma of patients with diabetic retinopathy(DR)to study the relationship between miR-1470 and diabetic retinopathy,and to verify the function of miR-1470 in diabetic retinopathy,to detect miR-1470 target genes,and to investigate the mechanism of miR-1470 affecting diabetic retinopathy.?Methods ?(1)Collect 5 ml of venous blood from DR patients,Diabetes Mellitus(DM)patients and normal subjects,extract plasma total RNA,and purify.The differentially expressed miRNAs in DR patients were screened by microarray.The expression of miR-1470 in plasma of DR patients was detected by RT-PCR.The target genes of miR-1470 were predicted and screened by miRWalk database and literature review.(2)The expression of miR-1470 in human retinal endothelial cells(HREC)was detected by RT-PCR.The expression of epidermal growth factor receptor(EGFR)was detected by Western Blot.Establish HREC miR-1470 high expression model,and cck-8 was used to detect the HREC proliferation ability,flow cytometry was used to detect the apoptosis of HREC.Scratch test was used to detect the migration ability of HREC.Western Blot was used to detect the expression of EGFR and Luciferase reporter assay was used to predict EGFR targets.(3)Predicting the long-noncoding RNA(LncRNA)binding to the miR-1470 locus was through the DIANA tool database.The expression of KCNQ1OT1 in HREC was detected by RT-PCR.The low expression model of HREC KCNQ1OT1 was established and the expression of miR-1470 was detected by RT-PCR,and the expression of EGFR was detected by Western Blot.?Results?(1)The results of gene chip detection showed that there were 195 miRNAs with differential expression in the DR group compared with the DM group and thenormal group.Among them,155 miRNAs were up-regulated and 40 miRNAs were down-regulated.The expression of miR-1470 in DR group was down-regulated 7.95 times compared with DM group and 12.31 times lower than that in normal group.RT-PCR validation results were consistent with the results of gene chip detection.The expression of miR-1470 in the DR group was(0.33±0.02)times that of the normal group and(0.42±0.04)times that of the DM group.There was a significant difference in the expression of miR-1470 among the three groups(F=63.486,P<0.05).Multiple comparison analysis showed that miR-1470 expression in the DR group was significantly decreased compared with the DM group and the normal group,and the difference was statistically significant(P<0.05).Compared with the normal group,miR-1470 expression in the DM group was reduced,but the difference was not statistically significant(P> 0.05).The miRWalk database predicts that EGFR is a target gene for miR-1470.(2)RT-PCR showed that the expression of miR-1470 in high glucose group was significantly lower than that in normal group(t=13.96,p<0.05).Western blot results showed that the expression of EGFR in the high glucose group was significantly higher than that in the normal group(t=37.97,p<0.05).RT-PCR results showed that the expression of miR-1470 in the blank control group,negative control group,and miR-1470 high expression group was significantly different(F=1417,P<0.05).Compared with the blank control group and the negative control group,the expression of miR-1470 in the high miR-1470 group was significantly increased(P<0.05).There was no significant difference in the expression of miR-1470 between the negative control group and the blank control group(P>0.05).The results of CCK-8 showed that the cell proliferation viability of the blank control group,negative control group,and miR-1470 high expression group had statistically significant difference(F=254,P<0.05).Compared with the blank control group and the negative control group,the cell proliferation activity of themiR-1470 high expression group was significantly lower(P<0.05).There was no significant difference in cell proliferation viability between the negative control group and the blank control group(P>0.05).The result of flow cytometry showed that the apoptosis rate of blank control group,negative control group and miR-1470 high expression group had statistical significance(F=2306,P<0.05).Compared with the blank control group and the negative control group,the apoptosis rate of the miR-1470 high expression group was significantly increased(P<0.05).There was no significant difference in apoptosis rate between the negative control group and the blank control group(P>0.05).The cell scratch test showed that the percentage of cell healing in the blank control group,negative control group,and miR-1470 high expression group had statistical significance(F=21.98,P<0.05).Compared with the blank control group and the negative control group,the healing percentage of the cells with high miR-1470 expression was significantly reduced(P<0.05).There was no significant difference in cell healing percentage between the negative control group and the blank control group(P>0.05).Western blot results showed that the expression of EGFR in the blank control group,negative control group,and miR-1470 high expression group had statistical significance(F=263.3,P<0.05).Compared with the blank control group and the negative control group,the expression of EGFR was significantly reduced(P<0.05).There was no significant difference in the expression of EGFR between the negative control group and the blank control group(P>0.05).Luciferase reporter gene results showed that relative luciferase activity in the EGFR group was significantly lower than in other groups.(3)DIANA tools predict that KCNQ1OT1 can bind to miR-1470 binding site.RT-PCR showed that the expression of KCNQ1OT1 in high glucose group was significantly higher than that in normal group(t=19.09,p<0.05).RT-PCR results showed that the expression of KCNQ1OT1 was significantly different between the blank control group,negativecontrol group,and KCNQ1OT1 low expression group(F=194.6,P<0.05).Compared with the blank control group and the negative control group,the expression level of KCNQ1OT1 in the KCNQ1OT1 low expression group was significantly lower(P<0.05).There was no significant difference in the expression of KCNQ1OT1 between the negative control group and the blank control group(P>0.05).RT-PCR results showed that the expression of miR-1470 in the blank control group,negative control group,and low expression group of KCNQ1OT1 was significantly different(F=248.1,P<0.05).Compared with the blank control group and the negative control group,the expression level of miR-1470 in the KCNQ1OT1 low expression group was significantly increased(P<0.05).There was no significant difference in the expression of miR-1470 between the negative control group and the blank control group(P>0.05).Western blot results showed that the relative expression levels of EGFR in the blank control group,negative control group,and KCNQ1OT1 low expression group were statistically significant(F=59.68,P<0.05).Compared with the blank control group and the negative control group,the relative expression of EGFR was significantly reduced(P<0.05).There was no significant difference in the relative expression of EGFR between the negative control group and the blank control group(P>0.05).? Conclusion ?(1)The plasma miR-1470 in patients with DR is significantly decreased,and EGFR may be the target gene for miR-1470.(2)Under high glucose conditions,miR-1470 expression was significantly reduced in HREC,and miR-1470 can significantly reduce the proliferation of HREC,significantly increase the apoptosis rate of HREC,significantly reduce the ability of HREC migration and healing,and bind to its target gene EGFR 3?-UTR and negative Regulates EGFR expression.(3)KCNQ1OT1 is significantly elevated in DR,and it can bind miR-1470 competitively with EGFR mRNA,thereby exerting its antagonistic effect onmiR-1470 inhibiting EGFR translation or transcription. |
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