Objective To observe the expression of miR-204 and 211 human embryonic stem cells (hESCs) differentiated into retinal pigment epithelial (RPE) cells. Methods RPE cells were derived from hESCs by natural differentiation method, and were identified. miRNA expression profiles and real-time polymerase chain reaction (RT-PCR) of miR-204 and 211 were generated from the following groups: hESCs, hESCs-derived cells containing pigmented foci, hESCs-derived RPE cells and human fetal RPE (hfRPE) cells. Results miRNA-204 was continuously upregulated throughout the entire differentiation process of hESCs to RPE cells. It increased 5.026 times in hESCs-derived cells containing pigmented foci compared to hfRPE cells; it was increased 3.337 times in hESCs-derived RPE cells compared to hESCs-derived cells containing pigmented foci; it increased 13.574 times in hfRPE cells compared to hESCs-derived RPE cells. miR-211 does not change during differentiation from hESC to RPE, but it increased 44.333 times in hESCderived RPE cells compared to hfRPE cells. miR-211 was the biggest difference in the miRNA expression pattern. In four cell types of hESCs, hESCs-derived cells containing pigmented foci, hESCs-derived RPE cells and hfRPE cells, RT-PCR showed the levels of miR-204 were 91.81plusmn;4.43, 2263.09plusmn;206.39, 5996.80plusmn;235.42, and 171676.45plusmn;999.82 respectively. miR-204 was significantly increased during the whole course (t=18.22, 20.66, 279.38;P＜0.001). The levels of miR-211 were 2.23plusmn;0.31, 129.33plusmn;3.75, 125.7592plusmn;4.78, and 16682.00plusmn;352.97 respectively. miR-211 was significantly increased from hESCs to cells containing pigmented foci and from hESCs-derived RPE cells to hfRPE (t=58.58, 81.24; P＜0.001). Conclusion There is a continuous change of miR-204 and 211 in differentiation of RPE cells from hESCs.
ObjectiveTo study morphological characteristics and microRNA (miR) expression profiling in a mouse model of oxygen-induced retinopathy (OIR). MethodsHealthy C57BL/6J female mice and pups were randomly divided into normal and OIR group at postnatal day 7 (P7). The normal group was raised in a conventional cage and exposed to room air for 10 days. The OIR group was raised in a sealed chamber and exposed to (75±2)% oxygen. The moms were alternated between the two groups every day to promote their survival under hyperoxia. The OIR group was returned to the room air at P12. At P17, mice from either group were retro-orbitally injected with high molecular weight fluorescein isothiocyanate-dextran (FITC-dextran), the eye balls were fixed in 4% paraformaldehyde, and the retinal whole mounts were prepared. The retinal vessels labeled with FITC-dextran were observed under a fluorescence microscope; the eye balls were also processed for paraffin sections and Hematoxylin and Eosin (H&E) staining. The cell nucleus in the newly-formed vessels beyond the inner limiting membrane was quantified. The miR was extracted from the eyes, reverse transcribed, and subjected to a customized miR array analysis. The real-time PCR was preformed to verify the results of the miR array. ResultsRetinal whole mounts labeled with FITC-dextran showed that the peripheral retinal microvessels in the OIR group were tortuous, disorganized with neovascular buds, and the avascular area was prominent in central retina. In contrast, the vessels were smooth, organized, and evenly distributed in the retinas of normal group. The percentage of avascular area in total retina area in OIR group (25.81±2.12)% was 4-fold that in normal group (6.57±3.6)% (P < 0.01, normal group vs OIR group). H & E staining showed that the number of the cell nuclei beyond inner limiting membrane was (28.41±4.01) in OIR retina, which was substantially higher than that (0.16±0.31) in normal retina (P < 0.01, normal group vs OIR group). More interestingly, the results of miR array showed that 21 out of the 80 miRs examined exhibited more than 1.5-fold changes at expression level. Among these 21 miRs, 9 were up-regulated, 12 were down-regulated; 4 miRs showed more than 3-fold expression changes, 3 were down-regulated and 1 was up-regulated. The expression of the 4 miRs was verified by real-time PCR. The expression trends of miR-3078, miR-140, miR-29b and miR-29c were consistent with those revealed by the miR array. MiR-3078 was significantly up-regulated (t=-2.380, P < 0.05. normal group vs OIR group), and the other 3 miRs were significantly down-regulated (t=2.638, 2.323, 2.415, P < 0.05. normal group vs OIR group). ConclusionsThe OIR mouse model has been established in our study. Differential expression of the microRNAs, including miR-3078, 140, 29b and 29c, was detected in normal and OIR mouse retinas. These miR expression changes may be associated with retinal neovascularization. These results would provide the new leads for further studying pathogenic mechanisms and therapeutic targets for neovascular retinopathy.
ObjectiveTo investigate the expression of miR-195 and the underlying molecular mechanisms of miR-195 regulating HMGB1 in diabetic retinopathy (DR). MethodsExtract 5 ml venous blood from DR patients, diabetes mellitus (DM) patients and normal subjects, then extract and perificate plasma total RNA. MicroRNA array and real time polymerase chain reaction (RT-PCR) was used to screen out miRNAs which were expressed with significant differences in the serum of patients with DR. Bioinformatics was employed to predict the miR-195 related to high mobility group box 1 (HMGB1) regulation. Next, miR-195 was down-regulated or up-regulated in umbilical vein endothelial cells through transfection of miR-195 inhibitor and miR-29b mimics respectively.Then we analyzed expression of HMGB1 mRNA and protein by RT-PCR and Western blot. ResultsMicroRNA array results showed the expression of miR-195 in DR group is decreased by 8.34 times and 11.47 times compared with DM group and the normal group. RT-PCR verification results conforms to the microRNA array results. Compared with the DM group (F=0.034, t=8.057) and the normal group (F=0.370, t=9.522), the expression of miR-195 in DR group were significantly reduced, the differences were statistically significant (P < 0.05). RT-PCR showed that the expression of HMGB1 mRNA was significantly decreased in up-regulation group, compared with blank (F=0.023, t=11.287) and negative control group (F=0.365, t=7.471), the difference was statistically significant (P < 0.05). The expression of HMGB1 mRNA was significantly increased in down-regulation group, compared with blank (F=0.053, t=10.871) and negative control group (F=0.492, t=6.883), the difference was statistically significant (P < 0.05). Western blot showed that the expression of HMGB1 protein was significantly decreased in up-regulation group, compared with blank (F=0.021, t=8.820) and negative control group (F=0.039, t=7.401), the difference was statistically significant (P < 0.05); and significantly increased in down-regulation group, compared with blank (F=0.186, t=10.092) and negative control group (F=0.017, t=12.923), the difference was statistically significant (P < 0.05). ConclusionMiR-195 can inhibit the expression of HMGB1, reduce the inflammation and angiogenesis, thereby delaying or inhibiting the occurrence and development of DR.
ObjectiveTo investigate the expression and mechanism of miR-1470 in plasma of diabetic retinopathy (DR) patients.MethodsThirty patients with DR (DR group), 30 patients with diabetes (DM group) and 30 normal healthy subjects (normal group) were enrolled in the study. Three groups of subjects were taken 5 ml of venous blood, and total plasma RNA was extracted and purified. The differentially expressed miRNAs in the plasma of DR patients were screened by gene chip, and the results of gene chip detection were verified by reverse transcription polymerase chain reaction (RT-PCR). Bioinformatics was used to predict potential target genes for miRNA regulation, and miR-1470 and its target gene epidermal growth factor receptor (EGFR) were screened. Human retinal microvascular endothelial cells (hREC) were divided into normal group (sugar concentration 5.5 mmol/L) and high glucose group (sugar concentration 25.0 mmol/L). hREC was transfected into miR-1470 mimics to establish a miR-1470 high expression cell model, which was divided into blank control group, high expression group and negative control group. The expression of miR-1470 was detected by RT-PCR. The expression of EGFR protein was detected by Western blot. The measurement data of the two groups were compared using the independent sample t test. The comparison of the measurement data between the two groups was analyzed by ANOVA. The comparison between the measurement data of the groups was compared by multiple comparisons.ResultsThe results of RT-PCR were consistent with those of the gene chip. The expression of miR-1470 in the plasma of the DR group, the DM group and the normal group was statistically significant (F=63.486, P=0.049). Compared with the DM group and the normal group, the expression of miR-1470 in the DR group was significantly decreased, and the difference was statistically significant (q=111.2, 73.9; P＜0.05). The expression of miR-1470 in hREC in the high glucose group was significantly lower than that in the normal group (t=42.082, P=0.015). The expression of EGFR protein in hREC of high glucose group was significantly higher than that of normal group (t=−39.939, P=0.016). The expression of miR-1470 (F=637.069, P=0.000) and EGFR (F=122.908, P=0.000) protein expression in hREC of blank control group, negative control group and high expression group were statistically significant . Compared with the blank control group and the negative control group, the expression of miR-1470 in hREC of high expression group was significantly increased (q=329.7, 328.8; P＜0.05), and the expression of EGFR protein was significantly decreased (q=242.5, 234.6; P＜0.05). There was no significant difference in the expression of miR-1470 and EGFR protein in hREC between the negative control group and the blank control group (q=1.5, 7.9; P＞0.05).ConclusionThe expression of miR-1470 in the plasma of patients with DR is significantly down-regulated, and the increase of EGFR expression may be related to it.
MiRNAs are stable small RNAs that are expressed abundantly in animals and plants. They can bind to the 3'-untranslated region of the target mRNA, and regulate its expression at the post-transcriptional level. The miRNAs’ abnormal expression and its following abnormal biological regulation are closely related to the occurrence and development of age-related macular degeneration (AMD), including inflammatory response, oxidative stress injury, phagocytosis dysfunction and abnormal angiogenesis. Since the dysregulation of miR-155, miR-125b and miR-34a seems to play a more important role in AMD, these microRNAs may be expected to become the new biomarkers and therapeutic targets for AMD.
ObjectiveTo analyze the expression of miRNA involved in regulating retinal neovascularizationin in retinal tissue of oxygen-induced retinopathy (OIR) mice.MethodsEighty healthy C57BL/6J mice were randomly divided into control group and OIR group at postnatal day 7(P7). Control group were not received any treatment and then exposed to room air. The OIR group was exposed to (75±2)% oxygen and then under room air at P12. Mice of all groups were euthanized at P17. Retinal neovasculation (RNV) was evaluated by counting the number of pre-retinal neovascular cells and analysing no perfusion area by immunofluorescent staining of the mouse retina.Total RNA was extracted from retinal tissue,and miRNA microarrays was performed to identify differentially expressed miRNA in the two groups. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed differential microRNA.ResultsCompared with the control group,the retinal neovascular tufts and the no perfusion area were both significantly smaller than those in OIR group. The number of pre-retinal neovascular cell nuclei in retinas from control group were obviously lower than those in the retinas from OIR group (t=9.025, P＜0.05). MiRNA microarray analysis showed that 54 miRNA in OIR group showed statistically different expression in control group, 47 miRNA were up-regulated and 7 miRNA were down-regulated. The results of PCR were consistent with the trend of microarray. In GO analysis, 1112 items were significantly different (P＜0.05), and 65 items were significantly different in KEGG analysis of expression profile (P＜0.05).ConclusionsThe miRNA expression in retinal tissue of OIR mice is different from that of normal mice, and these miRNA may be involved in the development of RNV. There are 54 miRNA expression differences in retinal tissue of OIR compared with normal mouse retinal tissue.
Age-related macular degeneration (AMD) is a multifactorial disease affected by environmental factors and genetic variation, which is a major cause of irreversible vision loss in the elderly. miRNA is a kind of endogenous non-coding RNA, which plays an important role in the pathogenesis of AMD, such as oxidative stress, pathological neovascularization and inflammation, by inhibiting or silencing the expression of transcription genes. miRNA has unique advantages in terms of ease synthesis, targeting and additive effect, a large number of experiments have proved the therapeutic potential of miRNA in AMD, which is expected to become a new method for the treatment of AMD in the future. Since the pathogenesis of AMD has not been fully elucidated, it is still necessary to continue to study the pathogenesis of AMD, the biological effects and mechanisms of various miRNA in the occurrence and development of AMD, and observe its therapeutic effects in AMD, so as to provide more effective options for the precise prevention and treatment of AMD.
ObjectiveTo observe the inhibitory effect of lentivirus (LV)-mediated miR-191 on the proliferation and angiogenesis of human retinal vascular endothelial cells (hREC) cultured in vitro.MethodsThe hREC cell lines were cultured in vitro and divided into control group, hypoxia group, LV-empty vector (LV-vector) group, and LV-miR-191 (LV-191) group. The LV-vector group and LV-191 group were transferred to the corresponding lentiviral vector respectively. Flow cytometry was used to detect cell transfection efficiency. Cell Counting Kit-8 (CCK-8) test was used to detect cell proliferation ability. Scarification test and invasion chamber (Transwell) test were used to detect cell migration ability. Matrigel test was used to detect cell lumen formation ability. Real-time quantitative polymerase chain reaction (qPCR) was used to detect the relative expression of miR-191 and relative mRNA expression of its downstream target genes p21, vascular endothelial growth factor (VEGF), cell division protein kinase (CDK) 6, cyclin-D1 (Cyclin D1). Independent sample t test was used for pairwise comparison. ResultsThe results of flow cytometry showed that the transfection efficiency of cells in the control group and the LV-191 group were 0.615% and 99.400%, respectively. The results of CCK-8, scarification, Transwell and Matrigel test showed that, compared with the control group, the number of cell proliferation (t=6.130, 4.606), the cell mobility (t=4.910, 6.702), the number of stained cells on the microporous membrane (t=7.244, 6.724) and the lumen formation ability cells (t=8.345, 9.859) were significantly increased in the hypoxia group and the LV-vector group (P<0.01), while the LV-191 group showed completely opposite performance (t=14.710, 6.245, 5.333, 5.892; P≤0.01). The qPCR test results showed that, compared with the control group and the LV-vector group, the relative expression of miR-191 mRNA in the cells of the LV-191 group was significantly up-regulated (t=44.110, 42.680), the relative expression of Cyclin D1 mRNA (t=29.940, 14.010) and CDK6 mRNA (t=15.200, 7.645) decreased significantly, and the difference were statistically significant (P<0.01); the relative expression of p21 mRNA increased, however, the difference was not statistically significant (t=2.013, 2.755; P>0.05). There was no significant difference in the relative expression of VEGF mRNA in the 4 groups of cells (F=0.966, P>0.05). ConclusionsLV-191 can inhibit the proliferation, migration and tubing of hREC by up-regulating p21 and down-regulating CDK6 and Cyclin D1.