Objective To investigate the effect of microRNA-27a (miR-27a) on the apoptosis of human lung adenocarcinoma cells A549 induced by lipopolysaccharide (LPS) by regulating the phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT) pathway, and its mechanism is discussed preliminarily. Methods The complementary binding sites of miR-27a and phosphatidylinositol-3 kinase catalytic subunit delta (PIK3CD) were analyzed by Starbase and verified by double luciferase. The A549 cells were divided into normal group, LPS group, LPS+miR-27a mimic negative control group, LPS+miR-27a mimic group, LPS+miR-27a mimic+PI3K activator group. In the LPS+miR-27a mimic negative control group, LPS+miR-27a mimic group and LPS+miR-27a mimic+PI3K activator group, the cells were transfected with miR-27a mimic negative control, miR-27a mimic and miR-27a mimic, respectively, and were cultured for 6 h. After that, the cells were cultured in complete medium for 24 h, and then, except for the normal group, the cells in the other groups were stimulated with 10 mg/L LPS for 24 h, and the PI3K activator 740 Y-P was added to the LPS+miR-27a mimic+PI3K activator group, and cells in normal group were cultured in complete medium for the same time. Real-time quantitative polymerase chain reaction was used to detect the expression level of miR-27a in cells; cell counting kit 8 was used to detect cell proliferation; Hoechst33342 staining and flow cytometry was used to detect apoptosis; autophagy of A549 cells was observed by transmission electron microscope; Western blot was used to detect the expression of PIK3CD, phosphorylated-AKT (p-AKT), B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax), cleaved caspase-3 and microtubule-associated protein 1 light chain 3 II (LC3II) protein. Results There was a binding site between miR-27a and PIK3CD, which was verified by double luciferase. Compared with those in normal group, the expression level of miR-27a, proliferation rate and protein expression level of Bcl-2 in LPS group and LPS+miR-27a mimic negative control group were lower (P<0.05), the apoptosis rate, protein expression levels of PIK3CD, p-AKT, Bax, cleaved caspase-3, LC3Ⅱ were higher (P<0.05); compared with those in LPS group and LPS+miR-27a mimic negative control group, the expression level of miR-27a, proliferation rate and protein expression level of Bcl-2 in LPS+miR-27a mimic group were higher (P<0.05), the apoptosis rate, protein expression levels of PIK3CD, p-AKT, Bax, cleaved caspase-3, LC3Ⅱ were lower (P<0.05); compared with those in LPS+miR-27a mimic group, the expression level of miR-27a and proliferation rate in LPS+miR-27a mimic+PI3K activator group were lower (P<0.05), the apoptosis rate, protein expression levels of PIK3CD, p-AKT, cleaved caspase-3, LC3Ⅱ were higher (P<0.05). The number of cells in the normal group was more, the cells were closely arranged, the nucleus size was uniform, and the organelle structure was normal; in LPS group and LPS+miR-27a mimic negative control group, cells became round, nuclei pyknosis, formed clumps, and showed multiple round autophagic vesicles of different sizes; the number of nuclear pyknotic cells in LPS+miR-27a mimic group decreased, and the number of nuclear pyknotic cells in LPS+miR-27a mimic+PI3K activator group increased compared with LPS+miR-27a mimic group, a small number of circular autophagic vesicles were observed, but the number was different. Conclusion Overexpression of miR-27a can inhibit PI3K/Akt pathway and reduce LPS induced apoptosis of human lung adenocarcinoma cells A549, which may be related to the reduction of autophagy.
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.
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 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.
Objective To observe the expression of miRNA in retinal tissue of mice with oxygen-induced retinopathy (OIR), and screen miRNAs related to p21 and retinal neovascularization (RNV) formation. MethodsA experimental study. Forty healthy 7-day-old C57BL/6J mice were randomly divided into normal group and OIR group, with 20 mice in each group. The oxygen induced RNV model was constructed in the OIR group, and no treatment was performed in the normal group. At the age of 17 days, the mice were killed and the RNV of mice was observed by retinal fluorescence; the nuclei of vascular endothelium that broke through the inner limiting membrane of retina were counted under light microscope. The retinal tissues were taken for miRNA chip analysis to detect the differentially expressed miRNAs between the normal group and the OIR group. The resulting differential miRNA target genes were subjected to enrichment analysis based on gene annotation (GO) and Kyoto Encyclopedia of genes and genomes (KEGG); miRNAs and pathways that may be related to p21 were screened through Targetscan, MiRanda and MicroT-CDs database alignment. Independent sample t-test was used for pairwise comparison between groups. ResultsCompared with the normal group, the area of nonperfusion area, RNV and the number of vascular endothelial nuclei that broke through the inner limiting membrane of the retina in the OIR group increased significantly, differences were statistically significant (t=18.800, 9.025; P＜0.05). Compared with the normal group, there were 54 miRNAs that were statistically differentially expressed in the OIR group, of which 47 were up-regulated and 7 were down-regulated. A total of 13 miRNAs related to p21 were screened from the alignment results of the three databases with the obtained differential miRNAs. According to the difference multiples, they were miR-7218-5p, miR-322-5p, miR-224-5p, miR-335-5p, miR-329-3p, miR-362-3p, miR-532-5p, miR-20b-5p, miR-20a-5p, miR-195a-5p, miR-423-5p, miR-497a-5p, and miR-129-5p. Differential miRNA target gene enrichment analysis yielded 1 112 go entries and 50 KEGG pathways, of which 50 go entries and 13 KEGG pathways were related to p21. Conclusion13 miRNAs related to p21 were screened out in the OIR model.
Objective To explore whether microRNA-203 (miR-203) targets and regulates the Toll-like receptor 4 (TLR4)/nuclear transcription factor kappa B (NF-κB)/nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) to protect alveolar epithelial cells from lipopolysaccharide (LPS)-induced apoptosis and inflammation injury. Methods The alveolar epithelial A549 cells were used as the research objects and divided into: Control group (normal culture), LPS group (LPS treatment), LPS+miR-NC mimics group (LPS treatment after transfection of miR-NC mimics), LPS+ miR-203 mimics group (LPS treatment after transfection of miR-203 mimics), LPS+miR-203 mimics+pcDNA group (LPS treatment after transfection of miR-203 mimics and pcDNA), LPS+miR-203 mimics+pcDNA-TLR4 group (LPS treatment after transfection of miR-203 mimics and pcDNA-TLR4). Dual luciferase reporter gene was used to detect the targeting relationship between miR-203 and TLR4; Real-time quantitative reverse transcription-polymerase chain reaction was used to detect the relative expression levels of miR-203 and TLR4 mRNA; enzyme-linked immunosorbent assay was used to measure the levels of tumor necrosis factor-α (TNF-α), interleukin (IL)-1β and IL-6; flow cytometry was used to detect the apoptosis rate of A549 cells; Western blot was used to detect the expression of B-cell lymphoma/leukemia-2 gene (Bcl-2) and Bcl-2 associated X protein (Bax), TLR4, NF-κB and NLRP3 proteins in A549 cells. Results There was a targeted regulation relationship between miR-203 and TLR4. Compared with the Control group, the expression of miR-203, TLR4 mRNA and protein, Bax, NF-κB, and NLRP3 proteins in A549 cells in the LPS group increased, the levels of TNF-α, IL-1β and IL-6 in the cell supernatant increased, the apoptosis rate increased, the level of Bcl-2 protein in cells decreased (P<0.05). Compared with the LPS+miR-NC mimics group, the expression of TLR4 mRNA and protein, Bax, NF-κB, and NLRP3 proteins in A549 cells in the LPS+miR-203 mimics group decreased, the levels of TNF-α, IL-1β and IL-6 in the cell supernatant decreased, the apoptosis rate decreased, the expression level of miR-203 and the level of Bcl-2 protein in cells increased (P<0.05). Compared with the LPS+miR-203 mimics+pcDNA group, the expression of miR-203, TLR4 mRNA and protein, Bax, NF-κB, and NLRP3 proteins in A549 cells in the LPS+miR-203 mimics+pcDNA-TLR4 group increased, the levels of TNF-α, IL-1β and IL-6 in the cell supernatant increased, the apoptosis rate increased, the expression level of miR-203 and the level of Bcl-2 protein in cells decreased (P<0.05). Conclusion MiR-203 can target TLR4/NF-κB/NLRP3 to protect alveolar epithelial cells from apoptosis and inflammation induced by LPS.
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.