ABSTRACT
Background@#More than ten genome-wide association studies have identified the significant association between the gap junction delta-2 (GJD2) gene and myopia. However, no functional studies have been performed to confirm that this gene is correlated with myopia. This study aimed to observe how this gene changed in mRNA and protein level in the form-deprivation myopia (FDM) animal model.@*Methods@#Four-week-old guinea pigs were randomly divided into two groups: control and FDM groups (n = 12 for each group). The right eyes of the FDM group were covered with opaque hemispherical plastic lenses for 3 weeks. For all the animals, refractive status, axial length (AL), and corneal radius of curvature were measured at baseline and 3 weeks later by streak retinoscope, A-scan ultrasonography, and keratometer, respectively. Retinal GJD2 mRNA expression and connexin 36 (Cx36) levels in FDM and control groups were measured by quantitative real-time PCR and Western blot analyses, respectively. Those results were compared using independent t test, Mann-Whitney U test, or paired t test. A significance level of P < 0.05 was used.@*Results@#Three weeks later, the FDM group (form-deprived eyes) showed about a myopic shift of approximately -6.75 (-7.94 to -6.31) D, while the control group remained hyperopic with only a shift of -0.50 (-0.75 to 0.25) D (Z=-3.38, P < 0.01). The AL increased by 0.74 (0.61–0.76) and 0.10 (0.05–0.21) mm in FDM and control groups, respectively (Z = -3.37, P < 0.01). The relative mRNA expression of GJD2 in the FDM group decreased 31.58% more than the control group (t = 11.44, P < 0.01). The relative protein expression of CX36 on the retina was lowered by 37.72% in form-deprivation eyes as compared to the controls (t = 17.74, P < 0.01).@*Conclusion@#Both the mRNA expression of GJD2 and Cx36 protein amount were significantly decreased in the retina of FDM guinea pigs. This indicates that Cx36 is involved in FDM development, providing compensating evidence for the results obtained from genome-wide association studies.
ABSTRACT
.
Subject(s)
Animals , Female , Pregnancy , Cell Communication/physiology , Diet, Protein-Restricted , Diabetes, Gestational/diet therapy , Intercellular Junctions/metabolism , Islets of Langerhans/metabolism , RNA, Messenger/metabolism , Analysis of Variance , Actins/metabolism , Adherens Junctions/metabolism , Blood Glucose/analysis , /metabolism , Connexins/metabolism , Diabetes, Gestational/prevention & control , Gap Junctions/metabolism , Glucose/administration & dosage , Insulin/metabolism , Insulin , Rats, Wistar , Real-Time Polymerase Chain Reaction , beta Catenin/metabolismABSTRACT
Objective To investigate the expression and association of Cx36 with ZO-1 in Cx36 transfected HeLa cells. Methods RT-PCR,PCR products cloning into PCR2.1TOPO vector,Cx36-pcDNA3 expression vector construction,cell culture,transient transfection using lipofectamine 2000,stable clone screening with G418,Western blotting,double immunofluorescence,and immunoprecipitation(IP) were used. Results Cx36-pcDNA3 expression vector was constructed and transfected into HeLa cells.Using homogenates from Cx36 transfected HeLa cells,Western blotting showed Cx36 band.By immunofluorescence microscopy,Cx36 transfected HeLa cells displayed punctate immunolabeling for Cx36 between cells.Irmmunolabeling for ZO-1 in these cells exhibited similar distribution to Cx36.By laser scanning confocal microscopy after double labeling with Cx36 and ZO-1 antibody revealed a high degree of Cx36 and ZO-1 colocalization at sites of cell-cell contact.Cx36 transfected HeLa cells were taken for IP with ZO-1 antibody,blots of IP protein probed with Cx36 antibody showed detectin of Cx36.Blots showed detection of ZO-1 after IP with Cx36 antibody from Cx36 transfected HeLa cells.Conclusion Cx36 was expressed in Cx36 transfected HeLa cells,Cx36 colocalizated and associated with ZO-1 in Cx36 transfected HeLa cells.