Running Title: miR-181a inhibits neovascularization
Keywords: miR-181a, anti-angiogenesis, ocular neovascularization, extracellular miRNAs (ex-miRNAs)
Abstract: (240 words)
Purpose: Excess angiogenesis or neovascularization plays a key role in the pathophysiology of several ocular diseases such as retinopathy of prematurity, diabetic retinopathy and exudative age-related macular degeneration. The expression of microRNA-181a (miR-181a) was found highly expressed in retina and choroidal tissues. This study aims to investigate the role of miR-181a in the regulation of ocular neovascularization in different pathophysiological conditions.
Methods: We performed the RNA sequence to identify the microRNAs components of anti-angiogenic lymphocyte-derived microparticles (LMPs). The effect of miR-181a on human retinal endothelial cells proliferation was assessed in vitro. The impact of miR-181a on angiogenesis was confirmed using in vitro angiogenesis assay, ex vivo choroidal explant and in vivo retinal neovascularization. The expression of major angiogenic factors was assessed by real-time qPCR.
Results: RNA sequence revealed that miR-181a is selectively enriched in LMPs. Importantly, the inhibition of miR-181a significantly abrogated the effect of LMPs on endothelial viability, but overexpression of miR-181a reduced endothelial cell viability in a dose-dependent manner. miR-181a strongly inhibited in vitro angiogenesis and ex vivo choroidal neovascularization. The strong anti-angiogenic effect of miR-181a was also displayed on the retinal neovascularization of the in vivo mouse model of oxygen-induced retinopathy. In keeping with its effect, several angiogenesis-related genes were dysregulated in the miR-181a overexpressed endothelial cells.
Conclusion: These data may open unexpected avenues for the development of miR-181a as a novel therapeutic strategy that would be particularly useful and relevant for the treatment of ocular neovascular diseases.
Excess angiogenesis or neovascularization plays a key role in the pathophysiology of several ocular diseases such as retinopathy of prematurity, diabetic retinopathy and choroidal neovascularization associated with age-related macular degeneration (AMD) 1. It is widely accepted that angiogenesis is determined by a relative balance between pro- and anti-angiogenic factors 2. The angiogenesis-related factors form a well-coordinated and functional network of molecules affecting the angiogenesis process, thus an effective therapy may require targeting multiple components of the angiogenic pathway. Endothelial cells are involved in many aspects of vascular biology, producing different factors that regulate cell adhesion, cell proliferation, and vascular tone 3. The signaling of vascular endothelial growth factor (VEGF) is one of the most potent pathways and is almost exclusively found in endothelial cells. Importantly, a large number of microRNAs (miRNAs) are responsible for angiogenesis and are expressed in endothelial cells 4-6. MiRNAs are a group of endogenous small non-coding regulatory RNAs (~ 22 nucleotides) silencing their target genes at the post-transcriptional level7, 8. Each miRNA regulates the expression of multiple protein-coding genes and therefore miRNA-based therapy provides the rationale basis for an effective anti-angiogenic treatment 9.
The extracellular microvesicles (EVs) are biologic effectors to influence various physiological and pathological functions of recipient cells 10-12. We have demonstrated that EVs derived from apoptotic human T-lymphocytes (LMPs) possess strong anti-angiogenic properties in in vivo corneal neovascularization, tumor neovascularization 13, 14 and limit neovascularization of oxygen-induced retinopathy (OIR) and neovascularization in a laser-induced murine choroidal neovacularization (CNV) model 13-18. Nonetheless, the potent anti-angiogenic components of LMPs have not been well explored. Selective disposal of some miRNAs in EVs has been suggested to mediate both short-range and distant communication between various cells, and could impact diverse physiological and pathological processes 19-21. The main goal of this study is to investigate the miRNAs that mediate the anti-angiogenic effect of LMPs.
Human CEM T cells were purchased from (ATCC) and grown in X-VIVO medium (Cambrex). Human retinal endothelial cells (HREC) were obtained from Applied Cell Biology Research Institute and cultured as recommended. Human umbilical vein endothelial cell lines (HUVEC) were purchased from American Type Culture Collection (ATCC), and were maintained according to standard procedures.
LMPs were generated as described previously 13. Briefly, CEM T cells were treated with 0.5 μg/mL actinomycin D (Sigma-Aldrich) for 24 hours. A supernatant was obtained by centrifugation at 750 g for 15 min, then 1500 g for 5 min to remove cells and large debris. LMPs from the supernatant were washed after 3 centrifugation steps (50 min at 12000 g) and recovered in PBS. Washing medium from the last supernatant was used as a control. LMPs were characterized with annexin V (BD bioscience) staining by fluorescence-activated cell soring (FACS) analysis. The concentrations of LMPs were determined using the Bio-Rad protein assay.
RNA isolation from LMPs
RNAs were isolated from LMPs using the miRNeasy Mini Kit (Qiagen, Mississauga, Canada) according to the manufacturer’s protocol. Quality Threshold and the concentrations were determined using NanoDrop ND-1000 (NanoDrop Technologies, Wilmington, DE). High RNA quality of isolated RNA was confirmed for all samples using the BioAnalyzer 2100 (Agilent Technologies, Santa Clara, CA).
Expression profiling—Illumina BeadChips
Expression profiling of RNAs in LMPs with three biological replicates, was performed using the Illumina MiSeqBeadChips (Illumina Inc. San Diego, CA) where 200ng of total RNA was processed according to the protocol supplied by Illumina. The percentage of reads that fall on miRNA, rRNA, lincRNA, protein coding transcripts and pseudogenes was computerized based on gencode v19 annotations, and the features found in gencode were quantified using Cufflinks. Following read trimming and alignment, counts for all samples were extracted using HTSeq-count and the mirbase definition of known miRNAs (IRIC’s Genomics Core Facility, University of Montreal).
Human retinal endothelial cells (HREC) were transfected with 20nM of miR-181a inhibitor (hsa-miR-181a-5p inhibitor, Thermo Fisher Scientific) via liposomes CLS-3 (8 uM, a gift from Dr. Jeanne Leblond-Chain, University of Montreal) before the incubation with LMPs (10μg/ml). After 48 hours treatment, cell proliferation was evaluated by [3H]-thymidine incorporation assay as we described previously 13.
In a different experiment, endothelial cells were transfected with indicated concentrations of mirVana™ miRNA mimics (10, 25, 40 and 50 nM) for 48 hours. The mimics of miR-181a-5p (hsa-181a-5p) and miRscr (negative control #1) were purchased from Thermo Fisher Scientific. The cells were subjected to a proliferation assay.
Cells were seeded in 96-well plate. The next day, cells were exposed to Lenti-control (ABM) or Lenti-miR181a-5p (ABM) at MOI of 10 in the presence of polybrene (Sigma) at 8μg/ml or staurosporine (Cat. No.S4400, Sigma) as positive control in the presence of the RealTime-GloAnnexin V Apoptosis Assay Reagent (Promega) according to the protocol. The plate was incubated at 37°C with 5% CO2 and luminescence was collected kinetically with Clariostar (BMG Labtech) plate reader. The relative luminescence unit was presented.
Endothelial cell tube formation assay
The endothelial cell tube formation assay was performed as described 22. The mimic of hsa-miR-181a-5p (mirVana® miRNA mimic), and mirVana™ miRNA mimic, Negative Control were purchased from Thermo Fisher Scientific. HREC transfected with 50 nM mimic of miR-181a-5p or miRscr. 24 hours later, cells were seeded on MatrigelTM (BD Biosciences). The images were taken after 17-hour seeding using fluorescence microscopy (Eclipse E800, Nikon Corp).
Treatment of choroidal explants and measurement of neovascularization
The RPE-removed choroidal explants were prepared according to our previously described procedure 16, 17. Human eyes were from four donors, obtained from the Eye Bank of Canada. The study approval of the human clinical protocol and informed consent was obtained from CHU Sainte-Justine ethics committee, Ref # 3949, and our research adhered to the tenets of the Declaration of Helsinki. The choroid was sliced into 1~2-mm sections and placed in growth factor–reduced basement membrane matrix. Choroidal explants were cultured at 37°C in 5% CO2 for 5 days. The culture medium was changed on day 6 and explants were transduced with lenti-control and lenti-miR-181a-5p (106 U/ml) for 72 hours. Photographs of individual explants were taken at the end of treatment using an Axiovert 200M inverted microscope (Zeiss). The neovessel areas were determined using Image-Pro Plus software and presented as percentage of control (set as 100%).
Oxygen-induced retinopathy mouse model (OIR) and retina NV quantification
All animal experiments were performed according to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the Animal Care Committee of CHU Sainte-Justine (Montreal, QC, Canada). The OIR model is generated based on the established method 23. Briefly, newborn litters of C57BL/6 mice are placed in 75% oxygen from postnatal day (P) 7 until P12 and returned to room air. Retinal NV occurs between P12 and P17, triggering OIR. P12 OIR mice were randomly divided into 2 groups and each group (n=4 mice) and received intravitreal injections of 1 µL of1010U/ml of lenti-miR-181a-5p in one eye and lenti-control in the contralateral respectively. At P17, retinal flatmounts were prepared as described previously 24. To quantify the retinal neovascularization, vessels in flat-mounted retinas were stained with red fluorescent isolectin B4. The images of retinal flatmounts were taken and the area of neovascular tufts were measured as described25. Neovascularizations were quantified in a blind fashion by comparing the number of pixels in the neovascular tuft areas with the total number of pixels in the retina using the SWIFT-NV method 24, 26.
Quantitative RT-PCR and PCR array
Total RNA was extracted from HREC cells using an RNA extraction kit (Qiagen, Mississauga, ON). DNase-treated RNA was then converted into cDNA using M-MLV reverse transcriptase (Invitrogen). Quantitative analysis of gene expression was performed on a stratagene Mx3000p sequence detection system with SYBR Green Master Mix Kit (BioRad). Each sample was analyzed in triplicate and threshold cycle numbers were averaged. Gene expression was normalized to β-actin, and the percentage of reduction was calculated according to a previously described formula 27. PCR primers targeting humanMAPK1, Bcl-2, VEGF and β-actin were synthesized by Alpha DNA (Montreal, Quebec, Canada) based on the following sequences: MAPK1: forward 5′-GGCCCCTGAAAGAATAAACCC-3′; reverse 5′-CGAAGGATGGCCAACTCAATC-3′; Bcl-2: forward 5′-GGTGAACTGGGGGAGGATTG-3′, reverse 5′-GTGCCGGTTCAGGTACTCAG-3′; VEGF: forward 5′-CTACCTCCACCATGCCAAGT-3′; reverse 5′-GCAGTAGCTGCGCTGATAGA-3′; β-actin: forward 5’-CTGCGGCATTCACGAAACTAC-3’, reverse 5’-ATCTCTTTCTGCATCCTGTCCG-3’.
All experiments were repeated at least 3 times. Values are presented as means ± SEM. Data were analyzed by one-way ANOVA followed by post-hoc Bonferroni tests for comparison among means. Statistical significance was set at P < 0.05.
1. Inhibition of miR-181a significantly attenuated the effect of LMPs on HREC cell proliferation.
We have previously reported that LMPs exert a strong inhibitory effect on proliferation of endothelial and cancer cells 13, 15, 28. We have also demonstrated that LMPs significantly reduced the proliferation of human retinal endothelial cell (HREC) in a dose-dependent manner 15. As a first step in identifying miRNAs that are enriched in LMPs, we isolated the total RNAs from LMPs and performed RNAs sequence. The resulting data suggested that miRNAs are selectively incorporated into LMPs in which miR-181a is one of the most abundant miRNAs (Table 1). Thus, we speculated that miR-181a may play a role in mediating the effect of LMPs on endothelial cells. To prove this hypothesis, we used the inhibitor of miR-181a to block the activity of miR-181a when HREC cells were incubated with LMPs. The results of cell proliferation assay suggested that the inhibitor of miR-181a significantly, not dramatically, attenuated the effect of LMPs (Figure 1). The same phenomenon was observed in the proliferation assay of human umbilical vein endothelial cells (HUVECs) (Supplementary FigureS1).
Table 1. The read counts of most abundant miRNAs in LMPs. A table of small section from an Excel spread-sheet summarizing the analysis results of most abundant miRNA expression in LMPs.
|LMPs 1||LMPs 2||LMPs 3||mean|
2. miR-181a inhibited endothelial cell proliferation.
The synthetic mimic of miR-181a-5p was used to test anti-proliferative activity in HREC. The miR-181a-5p inhibited cell proliferation in a dose-dependent manner, producing up to 50% inhibition compared to a negative control miRNA (miRscr) (Figure 2A). miR-181a-5p was also overexpressed in the human umbilical vein endothelial cell (HUVEC) with lentiviral vector (lenti-miR-181a-5p), and cell growth was assessed by MTT assay. The cell growth of the HUVEC cells infected with lenti-miR-181a-5p was reduced in a dose-dependent manner (Figure 2B). Given the significant reduced cell growth by miR-181a, it is plausible to question whether miR-181a-5p affects cell apoptosis. To address this question, we performed the cell apoptosis assay on the HREC cells overexpressing miR-181a by lentiviral vector. The lenti-miR-181-5p at the MOI of 10 did not significantly induce HREC cell death (Figure 2C, P ˃0.05), although this dose of lenti-miR-181a-5p significantly suppressed cell growth.
3. miR-181a inhibited angiogenesis in vitro.
We performed an endothelial cell tube formation assay to assess the anti-angiogenic effect of miR-181a-5p in vitro. HREC transfected with mimic of miR-181a-5p showed dramatically decreased tube formation: a 70% decrease, compared to HREC transfected with miRscr mimic (Figure 3A, 3B).
4. miR-181a decreases neovascularization of human choroidal explants.
We investigated the effects of miR-181a on neovessel sprouting from cultured human choroidal explants. The human choroidal explants were transduced with lenti-control and lenti-miR-181a-5p. The neovascularized areas were strongly suppressed by miR-181a, with a reduction of 84.1±6.6%, compared to the control group (Figure. 4A, 4B).
5. miR-181a inhibits retinal neovascularization in the mouse model of oxygen-induced retinopathy.
To extend our investigation of the in vivo effect of miR-181a, we used an oxygen-induced retinopathy mouse model (OIR, a well-established animal model of ischemia-induced retinal neovascularization). Along with the in vitro and ex vivo effects, intravitreal injections of lenti-miR-181a-5p significantly decreased the retinal neovascularization areas, by 78% compared to the lenti-control group (Figure 5).
6. miR-181a altered the expression of angiogenic factors in endothelial cells.
To better understand how miR-181a produces its anti-angiogenic effect in endothelial cells, we performed quantitative PCR to analyze the expression of the target genes of miR-181a-5p in the HREC cell transduced with lenti-miR-181a-5p. These genes were selected based on the published papers 29-31 and database of TargetScanHuamn 7.1 (http://www.targetscan.org/), miRTarBase (http://mirtarbase.mbc.nctu.edu.tw/). They are involved in endothelial biological processes associated with angiogenesis, such as the cell cycle, cell migration, cell growth and proliferation 31. They are the mitogen-activated protein kinase 1 (MAPK1) 30, B-cell lymphoma 2 (Bcl2) 29, and VEGF 31. Compared to those in HREC cells transduced with lenti-control, the mRNA levels of these genes were all significantly reduced by miR-181a-5p (Figure 6).
There is mounting evidence that extracellular microvesicles (EVs) provide a means of intercellular communication both in physiological and pathological conditions, by local and systemic intercellular exchange of biological information 32. LMPs are membrane-derived EVs derived from human apoptotic T cells 13. The strong anti-angiogenic effect of LMPs has been demonstrated in vitro, ex vivo, as well as in several in vivo models 13-15. It is well documented that EVs harbour a concentrated set of phospholipids, cytokines, proteins, RNAs, DNA, etc., and can influence diverse biological functions 33. Since the discovery of miRNAs secreting within EVs (ex-miRNAs) in 2008, ex-miRNAs have been of interest to molecular biologists 19, 20. Our RNA sequence analysis revealed for the first time that miR-181a is one of the most abundant ex-miRNAs in the LMPs. This finding is supported by the fact that miR-181a was found highly expressed in the thymus, the primary lymphoid organ where maturation of T lymphocytes occurs in the early stages of T-cell differentiation 20, 34-38. Even though enriched in LMPs, miR-181a is one of the hundreds miRNAs expressed in LMPs. The inhibition of miRNA-181a caused only partial attenuation of the effect of LMPs (Figure 1), which suggested that miR-181a may not be the only active component. LMPs are heterogenic components containing lipids, proteins, DNAs and RNAs in addition to miRNAs 33. The strong anti-angiogenic effects of LMPs may resulted from the synergistic effects of many different components in LMPs, thus more in-depth studies are needed to explore the other active factors.
The members of the miR-181 family are evolutionarily conserved across almost all vertebrates, suggesting their functional importance 35, 39. Numerous studies have reported the involvement of miR-181a in important cell functions such as growth, proliferation, death, survival, maintenance, vascular cell signaling and blood vessel formation 9. Recently, the roles of miR-181a in the regulation of endothelial cell function, in vascular development, and in tumor angiogenesis have been studied in several in vitro and in vivo models 40-44. Nonetheless, the contradictory roles of miR-181a in modulating angiogenesis have been reported. Terek’s group reported that miR-181a is overexpressed in chondrosarcoma by hypoxia and VEGF 43, and miR-181a promotes tumor angiogenesis through directly targeting G-protein signaling 16 and consequently increasing CXC chemokine receptor 4 signaling 44. Conversely, the anti-angiogenic property of miR-181a was identified from 2 independent studies. First, Eom et al. found that miR-181a may inhibit mouse endothelial cells through negatively regulating VEGF receptor signaling 45, and then Li et al. demonstrated that ectopic miR-181a reduced in vivo angiogenesis via reduction of matrix metalloproteinase-14 expression in aggressive breast cancer cell lines 46. These controversial findings may suggest that the anti-angiogenesis effect of miR-181a is cell- or tissue-specific.
Ocular angiogenesis is a major cause of many ocular diseases and blindness. It is a significant contributing factor in diabetic retinopathy, exudative AMD, corneal neovascularization, retinopathy of prematurity, neovascular glaucoma, etc. The ocular expression of miR-181a has been studied in the mouse and in the human eye. In the mouse, miR-181a was identified as strongly expressed in the retina 47; in the human eye, miR-181a was abundantly expressed in the retina and RPE/choroid tissues 48. One in vitro study suggested that hypoxia increased the expression of miR-181a in choroidal endothelial cells 49. However, the role of miR-181a in the ocular pathophysiological angiogenesis has not been verified. Our current data generated from in vitro, ex vivo (choroidal neovascularization), and in vivo retinal neovascularization model (OIR), strongly support the anti-angiogenic role of miR-181a (Figures 2-6). Of special note, we observed that the concentration is critical for the anti-proliferation effect of miR-181a on endothelial cells, because the high dose of miR-181a (≥ 100nM of miR-181a-5p mimic) lost its effect on endothelial cell growth in vitro. Instead, endothelial cell growth was slightly increased, although not significant (p˃0.05 vs. control) (Appendix Figure S2). Similar results were also observed in miR-181a treated HUVEC cells when the concentration of lenti-miR-181a is high (MOI ≥100) (data not shown).
In pathological conditions, there is an imbalance of proangiogenic and anti-angiogenic factors secreted by retinal endothelial cells, and the over-expression of VEGF plays an important role in the pathogenesis of ocular angiogenesis 50. Recent studies have shown a reciprocity relationship between the angiogenic activity of VEGF and Bcl2; the latter is an anti-oxidant and anti-apoptotic resident mitochondrial protein 51. Nor et al. demonstrated that VEGF-mediated angiogenesis is associated with enhanced endothelial cell survival and induction of Bcl-2 expression 52. Moreover, Biroccio et al. reported that Bcl-2 enhanced VEGF expression and neovascularization in vivo 53. In the eyes, Bcl-2 expression in the endothelium plays a significant role during postnatal retinal vascularization 54. Bcl-2 deficiency attenuated ischemia-driven retinal neovascularization during OIR and pathological choroidal neovascularization 54, 55. Therefore, modulation of Bcl–2 expression plays a central role during angiogenesis. Notably, miR-181a was found to play a direct role in controlling mitochondrial function by directly regulating expression of Bcl-2 42. During the whole senescence process of primary human umbilical vein endothelial cells (HUVEC), miR-181a was found highly expressed 56, but Bcl-2 is downregulated in the senescence HUVEC cells 57. Although the combination of TargetScan and miRNAmap software does not predict that VEGF is the putative target gene of miR-181a, a correlation between the overexpression of miR-181a and VEGF was reported 31. In keeping with this observation, we also showed that the expression of Bcl-2 and VEGF was significantly downregulated by overexpression of miR-181a in HREC cells (Figure 6).
In addition to Bcl2, direct targeting of miR-181a to MAPK1 (also named ERK5, or BMK1) was confirmed by luciferase reporter gene assays 30. MAPK1 is expressed in a variety of tissues and its transcript is abundant in heart, placenta, lung, kidney, skeletal muscle and endothelial cells 58, 59. It can be activated by a range of growth factors, cytokines and cellular stresses. MAPK1-deficient mice and targeted deletion of MAPK1 in an adult mice model suggested an important role of MAPK1 in controlling angiogenesis 60-62. Since VEGF functions as a potent activator for MAPK1 in endothelial cells, MAPK1 is likely responsible for transmitting VEGF-dependent anti-apoptotic signals 62. One study also indicated the role of MAPK1 signaling in diabetic angiopathy 63. Herein, we observed that in HREC cells, overexpression of miR-181a strongly inhibited MAPK1 expression, which indicated that the anti-angiogenic effect of miR-181a may have resulted from the interfering the MAPK1/VEGF signalling. Thus, it is possible that miR-181a modulation of MAPK1 signalling may present a therapeutic window for aberrant ocular neovascularization.
In conclusion, we demonstrated for the first time that miR-181a exerts a strong anti-neovascularization effect in ocular angiogenesis models; in addition, miR-181a specifically targeted a set of angiogenic and cell growth-related genes. These data suggest that miR-181a may be developed as a new therapeutic strategy for treating ocular angiogenesis-related diseases.
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Figure 1. Inhibition of miR-181a significantly attenuated the effect of LMPs on HREC cell proliferation. 50nM of miR-181a inhibitor were transfected into human retinal endothelial cells (HREC) via liposomes CLS-3 before incubation with LMPs (10μg/ml). After 48 hours of treatment, cell proliferation was assessed and values were presented as percentages of control. ***p<0.01 vs. CTL, ≠p<0.05 vs. LMPs.
Figure 2. miR-181a inhibited endothelial cell proliferation and cell growth. A. Human retinal endothelial cells (HREC) were transfected with mimic of miR-181a-5p at indicated concentrations for 48 hours using lipofectamine 2000. Cell proliferation was assessed and values were normalized to cell proliferation of control cells and plotted as mean ± SE. (***p<0.01 vs. miRscr). B. miR-181a-5p was overexpressed in human umbilical vein endothelial cells (HUVEC) by lentiviral vector with different MOI (multiplicity of infection). Data were normalized to cell viability of control cells (infected with lenti-control and plotted as mean ± SE. (*p<0.05; **p<0.01 vs. lenti-control). C. HREC cell apoptosis was assessed by RealTime-GloAnnexin V Apoptosis Assay. (P ˃0.05 vs. lenti-control).
Figure 3. miR-181a inhibited angiogenesis in vitro. A. Representative images of tube formation assay after HREC transfected with mimic of 50 nMof miR-181a-5p or miRscr. 24 hours after the transfection, HREC cells were seeded on Matrigel. The images were taken after 17 hours of seeding using fluorescence microscopy. B. The tube formation was quantified by calculating the cumulative length of the tube of each image. Data are plotted as mean ± SE, *** p<0.001 vs. miRscr.
Figure 4. miR-181a decreased neovascularization of humanchoroidal explants. A. Representative images of human choroidal angiogenesis. The human choroidal explants were cultured in normal medium for 5 days for neovessel growth, and then tranduced with lenti-control and lenti-miR-181a-5p. The images were taken 3 days after lentivirus infections. B. The neovascularized areas in each condition were calculated and presented as a percentage of control (CTL, set as 100%). Data are plotted as mean ± SE, **p<0.01 vs. lenti-control.
Figure 5. miR-181a inhibited in vivo retinal neovascularization of oxygen-induced retinopathy (OIR) in mice. A. The images of retinal flatmounts from OIR mice were taken at postnatal day 17 (P17) after intravitreal injections at P12 with 1μl of 1010U/ml of lenti-control or lenti-miR-181a-5p. B. Retinal surface area covered by tufts (neovascularized area) was measured and quantified as a percentage of the entire retinal area, and these values were presented as relative to the lenti-control group, which was set as 100%. *P < 0.05 vs. lenti-control.
Figure 6. miR-181a suppressed the expression of MAPK1, BCL2, and VEGF in HREC cells. 50nM of miRscr and miR-181a-5p mimics were delivered into HREC cells respectively. 36 hours later the total RNAs were isolated and the indicated genes of interest were analyzed by quantitative RT-PCR. The values were presented as percentages of miRscr. *P<0.05, **P<0.01 vs. miRscr. MAPK1: mitogen-activated protein kinase 1; Bcl2: B-cell lymphoma 2; VEGF:vascular endothelial growth factor.