| Part 1 Expression level and significance of miR-127-5p in idiopathic macular epiretinal membrane vitreous bodyObjective: MiRNAs are differentially expressed and play an important role in the regulation of retinopathy,but there are few studies on the differential expression of miRNAs in idiopathic macular epiretinal membrane,but we speculate that there are also miRNAs in the idiopathic macular epiretinal membrane that have differential expression and important regulation.Therefore,in this experiment,we mainly use miRNAs chip technology to screen miRNAs with different expressions in the vitreous of patients with idiopathic macular epiretinal membrane and the control group.Methods: To find the miRNAs related to the idiopathic macular epiretinal membrane,we used microarray to detect the expression of miRNAs in the vitreous tissue of epimacular membrane and normal control vitreous tissue.At the same time,to further verify the test results and expand the clinical samples,qRT-PCR method was used to verify.Results: There were 61 miRNA genes differentially expressed in the vitreous tissues of idiopathic macular epiretinal membrane and normal control vitreous tissues,among which 24(39.3%)miRNAs differentially expressed were up-regulated in the vitreous tissues of idiopathic macular epiretinal membrane,37(60.7%)miRNAs differentially expressed were down regulated.The most obvious down-regulation were miR-127-5p,miR-486-3p,miR-660,miR-197,miR-425-5p,miR-518 b,miR-376 a,miR-145,miR-518 d,miR-191,miR-221 and miR-564.The most obvious up-regulation were miR-454,miR-302 a,miR-888,miR-302 c,miR-1305 and miR-519b-3p.miR-127-5p was most significantly down regulated in idiopathic macular epiretinal membrane vitreous body,In 37 cases of idiopathic macular epiretinal membrane vitreous and 20 cases of control vitreous,qRT-PCR experiment verified that the expression level of mir-127-5p in the vitreous of idiopathic macular epiretinal membrane patients was significantly down regulated.Conclusion: There are different miRNAs expression profiles in the vitreous of patients with idiopathic macular epiretinal membrane compared with the normal control group.MiR-127-5p,which has the largest down-regulation range,was confirmed by qRT-PCR.MiR-127-5p,which has the largest down-regulation range in the vitreous of patients with the idiopathic macular epiretinal membrane,can inhibit the cell growth of retinal müller cells after the increase of expression level Colonization ability.Part 2 MiR-127-5p regulates the proliferation,invasion,migration and apoptosis of retinal müller cells in ratsObjective: Increased expression of miR-127-5p could significantly inhibit rat retinal Müller cell proliferation.Therefore,we speculated that miR-127-5p might regulate other behaviors of Müller cells which played important role in the development of the epiretinal membrane.In the study,we explored the effects of increased expression of miR-127-5p on the proliferation,invasion,migration and apoptosis of Müller cells.Methods: MiR-127-5p mimics(10?M,30?M,100 ?M)were then transfected into rat retinal Müller cells to increase the expression of miR-127-5p.The proliferation ability of rat retinal Müller cells was detected by CCK-8 assay.The invasive ability of Müller cells was detected by transwell invasion assay,and the migration ability of Müller cells was detected by transwell migration assay.Flow cytometry was performed to detect Müller cell apoptosis.Results: MiR-127-5p mimics(10,30 and 100 ?M)could significantly inhibit the Müller cell proliferation after transfection into Müller cells.Increased expression of miR-RNA-127-5p also significantly inhibited retinal Müller cell invasion and migration ability.The increased expression of miRNA-127-5p significantly promoted retinal Müller cell apoptosis.Conclusion: Increased expression of miR-127-5p could inhibit the proliferation,invasion and migration of retinal Müller cells,and promote the apoptosis of retinal Müller cells.Part 3 The mechanism of miR-127-5p regulating müller cell activity in rat retinaObjective: Increased expression of miR-127-5p could inhibit the proliferation,invasion and migration of retinal Müller cells,and promote the apoptosis of retinal Müller cells.However,the regulatory mechanism of miR-127-5p on retinal Müller cells remained unclear.miRNAs usually could regulate physiological activity by regulating target genes.Therefore,we identified the target gene of miR-127-5p and explored its role in the regulation of the behavior of Müller cells by miR-127-5p in the study.Methods: Firstly,the target gene of miR-127-5p was screened by bioinformatics and validated by qRT-PCR.,The target gene and related signal pathway were analyzed by proteomics and bioinformatics.miR-127-5p mimics were first used to transfect rat retinal Müller cells to upregulate the expression level of miR-127-5p.Double luciferase assay was used to detect the targeting relationship between miR-127-5p and GLUL.Si-GLUL was used to decrease the expression levels of glutamine synthase in rat retinal Müller cells.The expression of RNA was detected by qRT-PCR and the expression of glutamine synthase protein was detected by Western Blot.The proliferation ability of rat retinal Müller cells was detected by CCK-8 assay.The invasive ability of Müller cells was detected by transwell invasion assay,and the migration ability of Müller cells was detected by transwell migration assay.Flow cytometry was performed to detect Müller cell apoptosis.Results: The results of protein mass spectrometry showed that miR-127-5p could significantly inhibit the expression of GLUL and related signaling pathways.GLUL gene was the target gene of miR-127-5p.and glutamine synthase was highly expressed in the vitreous of patients with the idiopathic anterior macular membrane.After knocking down the expression level of glutamine synthase by si-GLUL,the proliferation,invasion and migration ability of rats retinal Müller cells were significantly inhibited,and the apoptotic ability was increased.When the expression levels of miR-127-5p and glutamine synthase were up-regulated simultaneously,it was found that the decrease of proliferation,invasion and migration of Müller cells caused by the increase of miR-127-5p expression could be reversed by the up-regulated expression of glutamine synthase,and the decreased apoptotic ability of Müller cells caused by the increased expression of miR-127-5p could also be reversed by the up-regulation of glutamine synthase protein expression.Therefore,miR-127-5p could regulate the proliferation,invasion,migration and apoptosis of rat retinal Müller cells by targeting GLUL.Conclusion: GLUL was the target gene of miR-127-5p.The expression level of glutamine synthase encoded by GLUL was significantly up-regulated in the epiretinal membrane.miR-127-5p could regulate the proliferation,invasion,migration and apoptosis of rat retinal Müller cells by targeting GLUL.miR-127-5p and GLUL might be potential targets for the treatment or inhibition of epiretinal membrane development. |
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