Retinopathy of prematurity (ROP) is a blinding eye disease in children that is characterized by the formation of neovascularization in the retina; current treatments for this disease risk retinal damage and visual impairment. This study aimed to investigate the relationship between metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), miR-106a-5p, and matrix metalloproteinase 2 (MMP-2) in an oxygen-induced retinopathy mouse model and hypoxia-induced human retinal endothelial cells and to elucidate whether MALAT1 upregulates MMP-2 signaling by inhibiting miR-106a-5p. The role of this pathway in oxygen-induced murine retinopathy and its underlying mechanism were also investigated. MALAT1 inhibited the expression of miR-106a-5p and enhanced the expression of MMP-2, which in turn caused a series of pathological changes, such as the formation of new blood vessels in the retina. In addition, knockdown of MALAT1 can downregulate the expression of MMP-2 by sponging miR-106a-5p and inhibiting cell proliferation, migration, and tube-forming ability. In conclusion, our findings suggest that MALAT1 may contribute to the occurrence and development of ROP by inhibiting miR-106a-5p and increasing the expression of MMP-2, thus providing a new perspective for the targeted therapy of ROP.
Research Article
Long-chain non-coding RNA MALAT1 upregulates the MMP-2 signaling pathway by inhibiting miR-106a-5p in murine oxygen- induced retinopathy
https://doi.org/10.21203/rs.3.rs-2125896/v1
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retinopathy of prematurity
metastasis-associated lung adenocarcinoma transcript 1 (MALAT1)
retinal neovascularization
oxygen-induced retinopathy
matrix metalloproteinase 2 (MMP-2)
Retinopathy of prematurity (ROP) is a disorder of retinal vascular development in premature infants with low birth weight and a vascular proliferative retinal disease unique to premature infants. ROP is a complex disease process that is closely related to the lack of complete retinal vascular development in premature infants; however, its pathogenesis is unclear. At present, laser, condensation, anti-vascular endothelial growth factor, and other approaches are still the main treatment methods; however, the treatment process may damage the retinal tissue and cause visual impairment[1, 2].
Long non-coding RNAs (lncRNAs) play roles in a variety of biological processes and are important regulators of various biological processes including proliferation, differentiation, invasion and apoptosis[3]. The development of the retina also depends on its functions at different levels, including epigenetic, transcriptional, and post-transcriptional modifications[4]. A previous study found that lncRNA affects the development of the eyes of premature infants and plays an important role in the occurrence and development of ROP[5]; however, the role and mechanism of individual lncRNAs in ROP remains to be further studied.
The lncRNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is widely expressed in mammals, highly evolutionarily conserved, and involved in the occurrence and development of many human diseases. For example, it is involved in cardiovascular disease, endothelial cell dysfunction, tumor angiogenesis, and metastasis[6] Previous studies have reported that MALAT1 is involved in regulating the migration and proliferation of retinal pigment epithelium cells, thereby regulating the pathological process of proliferative vitreoretinopathy (PVR)[7]. MALAT1 can also compete and interfere with microRNAs (miRNAs) to build a competing endogenous RNA regulatory network to affect the expression of target gene mRNAs, thus taking part in angiogenesis and vascular remodeling of various vascular endothelial cells. This process mainly regulates angiogenesis by affecting the function of endothelial cells or endothelial progenitor cells (including cell proliferation, migration, differentiation, and aging) or by regulating the secretion of vascular endothelial growth factor (VEGF)[8–10]. In addition, Hou et al.[11] showed a regulatory relationship between MALAT1 and miR-140 and VEGF-A. MALAT1 knockout downregulates VEGF-A expression in hepatocellular carcinoma (HCC) and angiogenesis in human umbilical vein endothelial cells (HUVEC). MALAT1 can upregulate VEGF-A expression by inhibiting miR-140, thereby promoting angiogenesis in endothelial cells. MiRNAs are endogenous small non-coding RNA molecules, which could regulate the expression of its target mRNAs by binding to their 3′‐untranslated region (3′UTR)[12]. As a member of the neutral zinc protease family, MMP-2 have been implicated in a wide range of physiological and pathological processes, including wound healing, angiogenesis, inflammation, and tumor metastases[13, 14]. In addition, studies have shown that miR-106a was expressed differentially in diabetic retinopathy, and miR-106a overexpression showed protective effect in high glucose induced diabetes[15]. Bioinformatic analysis revealed that MALAT1 interacts with miR-106a-5p and matrix metalloproteinase 2 (MMP-2). In light of this background, we hypothesized that MALAT1 might be involved in retinal neovascularization (RNV). In order to verify this possibility, we investigated the role of MALAT1 in hypoxia-cultured human retinal endothelial cells (HRECs) and a model of oxygen-induced retinopathy (OIR) in mice. Hence, we further analyzed the impact of downregulating MALAT1 on miR-106a-5p/MMP2 regulation. These findings contribute to the search for a more effective clinical approach for ROP/RNV and provide insights into its pathogenesis.
2.1 Ethics statement
This study was approved by the Medical Ethics Committee of Shengjing Hospital, China Medical University (approval number: 2021PS536K). All animal experiments were conducted based on the principle of minimizing the number of animals and reducing the suffering of these animals.
2.2 Experimental Animals and Model Establishment
Healthy adult female and male C57/BL6J mice were purchased from Huafukang Company (Beijing, China) and used to breed young mice. The pups were randomly divided into 4 groups with 60 rats in each group: Control, OIR, OIR-NC, and OIR-mimics. Mice in the control group were fed by the mother mice under normal conditions. Mice in the OIR, OIR-NC, and OIR-mimic groups were exposed to 75% ± 2% oxygen on the 7th day after birth and returned to the normal environment on the 12th day after birth. Hyperoxia treatment resulted in vascular occlusion while the retina was still developing, and when the mice were returned to room air, it resulted in relative retinal hypoxia. The OIR-NC group and OIR-mimics group were injected with 0.5 µL of miR-106a-5p negative control reagent (miR-106a-5p NC mimics) + 0.5 µL into the vitreous cavity under isoflurane inhalation anesthesia on the 12th day, respectively. Animal transfection reagent + 0.5 µL PBS and 0.5 µL miR-106a-5p mimics (miR-106a-5p mimics) + 0.5 µL animal transfection reagent + 0.5 µL PBS. The sequences of the miR-106a-5p NC mimics were as follows: sense: 5'-UUCUCCGAACGUGUCACGUTT-3' and antisense: 5'-ACGUGACACGUUCGGAGAATT-3'. The sequences of the miR-106a-5p mimics were as follows: sense: 5'-AAAAGUGCUUACAGUGCAGGUAG-3' and antisense: 5'-ACCUGCACUGUAAGCACUUUUUU-3'. On days 14 and 17 after birth, the four groups of mice were selected to prepare follow-up experimental specimens for polymerase chain reaction (PCR) and other experiments.
2.3 Cell Culture and Transfection
HRECs were provided by Scientific Cell Research Laboratory, Carlsbad, California, USA. The 293T cell line was obtained from the Shanghai Cell Bank of the Chinese Academy of Sciences and identified using short tandem repeats. HRECs were cultured in RPMI 1640 medium (HyClone Laboratories, Logan, Texas, USA) and 10% fetal bovine serum (Biological Industries, Kibbutz Beit-Haemek, Israel), and 293T cells were cultured in Dulbecco’s modified DMEM medium (HyClone assay). Chamber, Logan, Texas, USA) and 10% fetal bovine serum (Biological Industries, Kibbutz Beit-Haemek, Israel) at 37°C and 5% carbon dioxide. All cells were passaged two to three times after resuscitation for subsequent experiments.
For MALAT1 knockdown experiments, the following groups were used: control group (Control group), cobalt chloride group (CoCl2 group), cobalt chloride + negative control RNA interference group (CoCl2 + NC group), and MALAT1-RNAi transfection group (CoCl2 + RNAi group).
For miR-106a-5p overexpression experiments, the groups were as follows: control group (Control group), cobalt chloride group (CoCl2 group), miR-106a-5p mimics negative control group (CoCl2 + NC group), and miR-106a-5p mimics group (CoCl2 + mimics group).
The cells in the experimental groups were treated with 200 µmol/L CoCl2 (Sigma Aldrich, St. Louis, MO, USA) for 24 h to simulate hypoxia. Cells were seeded at 70% density in six-well plates and transfected with MALAT1 RNAi (MALAT1-RNAi), negative control MALAT1-transfection of RNAi (RNAi-NC) (Genechem, Shanghai, China), miR-106a-5p mimics, and miR-106a-5p NC mimics (NC mimics) (GenePharma, Shanghai, China). The sequences used were as follows: MALAT1-RNAi: 5'-CTGCTATCTTAGCTGTCCTTA-3'; RNAi-NC:5'-TTCTCCGAACGTGTCACGT-3'; The sequence of the miR-106a-5p NC mimics was as follows: sense: 5'-UUCUCCGAACGUGUCACGUTT-3'; antisense: 5'-ACGUGACACGUUCGGAGAATT-3'. The sequence of the miR-106a-5p mimic was as follows: sense: 5'-AAAAGUGCUUACAGUGCAGGUAG-3'; antisense: 5'-ACCUGCACUGUAAGCACUUUUUU-3'.
2.4 RNA Extraction and Quantitative Real-Time Polymerase Chain Reaction
Total RNA was extracted from the retinal tissue of 14-day-old mice or cells from the aforementioned treatment groups using RNAiso Plus (Takara Biotech Co., Kyoto, Japan). The PrimeScript RT reagent Kit with gDNA Eraser (Takara, Japan) was used for reverse transcription, and TB Green PreMix Ex Taq II (Takara, Japan.) was used for quantitative PCR under the following thermocycling program: pre-degradation at 95°C for 30 s, followed by 40 cycles of 95°C for 3 s and 60°C for 30 s. The relative expression levels of MALAT1, miR-106a-5p, and MMP-2 were calculated by the 2−△△CT method. The mRNA and miRNA expression levels of the target genes were normalized to those of β-actin and U6, respectively. The specific primer sequences used for RT-PCR are listed in Table 1.
2.5 Retinal Vascular Staining
17-day-old mice were anesthetized with inhalation anesthesia so that to take out their eyeballs and then fixed for more than 4 h in 4% paraformaldehyde. The limbus was incised under a microscope, the retinal tissue was completely isolated after the anterior segment and vitreous were removed. Retinal tissue was cleaned with phosphate-buffered saline (PBS), stained with isolectin IB4 Alexa fluorochrome conjugate (Invitrogen, Waltham, MA, USA) dissolved in PBS containing 1% TritonX-100, and incubated overnight at 4°C. The retinal tissue was taken out the following day and washed with PBS. It was placed on a glass slide and under the microscope was cut into four pieces. Under a fluorescence microscope (Eclipse Ni; Nikon Inc., Melville, NY, USA), the slices were observed and photographed after being sealed with an anti-quencher. Retinal neovascular clusters and avascular areas were calculated using ImageJ software (NIH, USA).
2.6 Hematoxylin and Eosin Staining
The eyeballs of mice (17-day-old) were fixed overnight in 4% paraformaldehyde, and cut into 4 µm-thick sections after embedded in paraffin. After deparaffinization, the sections staining with hematoxylin-eosin (HE) and dehydrating, and finally sealed with neutral gum. We used a double-blind method to count the nuclei that penetrated the inner limiting membrane.
2.7 Immunohistochemistry
Using a streptavidin-biotin complex (SABC) kit (Boster Bio, Pleasanton, CA, USA), immunohistochemistry was performed. After deparaffinization, 3% citric acid repair solution was used to repair the sections, and dropwise incubation with 3% hydrogen peroxide for 10 min at room temperature was used to remove the endogenous oxidase. At 37 degrees Celsius, the sections were blocked with 5% goat serum for 30 minutes after being washed three times with PBS. Antibodies specific to rabbit IgG ((MMP-2, IL-1β, TNF-α, VEGF, HIF-1α, caspase-3, Bcl-2; 1:200; Immunoway Biotechnology Company, Plano, TX, USA)) were incubated with sections at 4°C overnight. PBS washed the sections three times the next day, treated dropwise with goat anti-rabbit IgG (1:200, Immunoway), incubated at 37°C for 30 min, and washed 3 times with PBS. SABC-POD was then added dropwise and the preparation was incubated at 37°C for 30 min. PBS was used instead of the primary antibody as the negative control. Immunohistochemical sections were stained with DAB for 60 s, photographed under a microscope after counterstaining with hematoxylin and mounting with neutral gum. Using Image-Pro Plus 6.0. to describe immunohistochemically positive cells by integrating optical density.
2.8 Western Blotting
Cells and retinal tissues were lysed with RIPA lysis buffer (Solarbio Life Science, Beijing, China) containing 1% PMSF to extract total protein, and proteins were quantified using the BCA Protein Quantiization Kit (Vazyme, Nanjing, China, Nanjing). The levels of retinal MMP-2, VEGF, hypoxia-inducible factor-1α (HIF-1α), interleukin (IL)-1β, tumor necrosis factor (TNF)α, Bcl-2, and caspase-3 expression were detected in the retina. After gel electrophoresis (80 V), the samples were transferred to polyvinylidene fluoride membranes. We then incubated the samples overnight at 4°C with the primary antibody (1:1000; Immunoway) after blocking with 5% bovine serum albumin. After 3 washes in TBST (0.05 M Tris, 0.15 M NaCl, pH 7.4 with 0.1% Tween-20), the samples were treated with horseradish peroxidase-labeled goat anti-rabbit IgG (H + L) Secondary antibody (1:2000, Immunoway) and incubated at 37°C for 1 hour. The western blotting results were obtained using chemiluminescence reagents (BeyoECL Plus, Shanghai, China) using a GE680 gel imager (GE Healthcare, Chicago, USA).
2.9 5-Ethynyl-20-deoxyuridine (EdU) Incorporation Assay
HRECs were seeded in a 12-well plate at a density of 3×104 cells/well and divided into the Control, CoCl2, Control-NC, and CoCl2 + RNAi groups, treated for 24 h, and then mixed with 5-ethynyl-2'- deoxyuridine (1× EdU) medium for 2 h. Next, the cells were treated with 4% paraformaldehyde for 20 min at room temperature, after which they were incubated with an osmotic agent (phosphate buffered saline containing 0.5% Triton X-100) for 10 min. Subsequently, the cells were stained with Azide 555 staining solution in the dark for 30 min, washed with detergent (3 times, 5 min each), and reacted with 1× Hoechst 33342 reaction solution at room temperature for 10 min in the dark. The following assays were performed using the EdU detection kit (BeyoClick™ EdU-555) according to the manufacturer's instructions. Nuclei were observed under a fluorescence microscope (Eclipse Ni; Nikon Inc., Melville, NY, USA). The cells whose nuclei were stained red were considered positive; cells whose nuclei were stained blue represented the total number. The quantitative data were expressed as the percentage of EdU-positive nuclei relative to the total number of nuclei counted.
2.10 Wound-Healing Assay
In a 6-well plate, HRECs were evenly seeded at a density of 1 x 106 cells, and each well was marked with three horizontal lines. The cells were treated and cultured overnight. After the cells had grown to more than 90% confluency, an even scratch was made with the tip of a 10 µL micropipette to the bottom horizontal line. A gentle wash of PBS was used to remove the damaged cell mass, and a photograph was taken, then cultured in serum-free medium for 48 h and photographed. Finally, we calculated the cell migration rate at 48 h as follows: cell migration rate = (area of the initial scratch − 48 h scratch area)/area of the initial scratch.
2.11 Transwell Assay
Transwell chambers (6.5 mm; Corning Glass Works, Corning, NY, USA) were used to detect in vitro cell migration in 24-well plates. Then, 500 µL of 1640 medium containing 10% fetal bovine serum (FBS) was added to the lower chamber. After transfection and CoCl2 treatment, 3×104 cells/mL were suspended in FBS-free RPMI 1640 medium, inoculated into the upper chamber, and cultured at 37°C and 5% CO2 for 24 h. The transwell chambers were fixed with 4% paraformaldehyde for 20 min, washed with PBS, stained with 0.1% crystal violet for 20 min, and washed with PBS. The cells that failed to invade were gently wiped off using a cotton ball. Stained cells were counted under an inverted fluorescence microscope (Eclipse Ni; Nikon Inc., Melville, NY, USA).
2.12 Flow Cytometry for Apoptosis
CoCl2 was added for 24 h after the HRECs were transfected. A trypsin of EDTA-free was used to digest cells, and after collecting and centrifuging the sample, the supernatant was removed and washed three times with PBS. An apoptosis detection kit (Vazyme Biotec Co., Nanjing, China) was used to stain the cells with Annexin-PE/7-AAD. According to the manufacturer's instructions, protected from light for 10 minutes at room temperature, and then obtain the proportion of apoptotic cells in each group by using a flow cytometer (BD, FACSCalibur, NJ, USA). Data analysis was performed using Cell Quest Pro software (Becton-Dickinson, CA, USA).
2.13 Tube Formation
A 50 µL volume of Matrigel (Corning Inc., Corning, NY, USA) diluted 2:1 with basal medium was evenly spread on the bottom of a pre-cooled (− 20°C) 96-well culture plate, and then the plate was placed at 37°C. The cells were then incubated in a cell incubator for 30 min, suspended in culture medium containing 10% fetal bovine serum, and seeded at 2×104 cells per well in Matrigel-coated 96-well plates. Six hours were incubated in microplates with cell suspensions from different treatment groups, tube formation was observed under an inverted microscope (ECLIPSE Ni-U, Nikon), and the number of nodes and grids was counted.
2.14 Dual Luciferase Reporter Gene Assay
Using the starbase bioinformatics website, we predicted the sites in hsa-miR-106a-5p that binding to MALAT1 and MMP-2. We designed and constructed the expression vector was, and hsa-miR-106a-binding to MALAT1 and MMP-2 was validated by using the dual-luciferase reporter gene. Firstly, seeded 293T cells into 96-well plates (5×103 cells/well),a firefly fluorescence expression vector and a Renilla fluorescence control plasmid were transfected when the cells reached approximately 70% confluency. After that, the experimental group was treated with a mimic of has-miR-106a-5P, while the control group was treated with the negative control vector. After 48 h of co-transfection, fluorescence was measured using a dual-luciferase detection kit (Promega Corporation, Madison, WI, USA).
2.15 Statistical Analysis
All values are expressed as the mean ± standard deviation (SD) of at least three independent experiments. Analysis of variance or Student's t-tests were performed using GraphPad Prism 8.0 software (GraphPad, USA). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0005. Statistical significance was set at P < 0.05.
3.1 MALAT1 Expression was Upregulated and miR-106a-5p was Downregulated in Oxygen-induced Retinopathy Mice
We examined the expression levels of MALAT1 and miR-106a-5p in the OIR retina in the Control and OIR groups by quantitative reverse transcription-polymerase chain reaction. According to the results, MALAT1 expression in the OIR group was significantly higher than that in the control group (Fig. 1A, ***P < 0.001), whereas miR-106a-5p expression was significantly lower than that in the control group (Fig. 1B, ****P < 0.0001).
3.2 Knockdown of MALAT1 Inhibits Hypoxia-induced Proliferation and Migration of Human Retinal Endothelial Cells
Hypoxic ischemic retinopathy is often characterized by the abnormal proliferation and migration of retinal endothelial cells. Therefore, it is important to evaluate the role of MALAT1 in cell proliferation and migration. First, we determined the relative expression of MALAT1 in the Control, CoCl2, CoCl2 + NC, and CoCl2 + RNAi groups (Fig. 2A). It was found that MALAT1 expression in CoCl2 group was significantly higher than that in Control group (***P < 0.001), and that in CoCl2 + RNAi group, MALAT1 expression was significantly lower than that in CoCl2 + NC group (**P < 0.01). These results suggest that hypoxia-induced increase in MALAT1 expression in HRECs can be downregulated by MALAT1-RNAi. To investigate the functional relevance of hypoxia-induced MALAT1 upregulation, we examined the effect of MALAT1 knockdown on the proliferation and migration of HRECs. The results showed that hypoxia induced cell proliferation while MALAT1 downregulation inhibited the proliferation of HRECs (Fig. 2B, C, *P < 0.05). The wound healing and migration experiments showed that compared with the Control group, the migration ability of the cells in the CoCl2 group was significantly enhanced (Fig. 2D-G, wound-healing experiment analysis **P < 0.01, transwell analysis ****P < 0.0001). The migration ability of cells in the CoCl2 + RNAi group was lower than that in the CoCl2 + NC group (wound-healing test analysis ***P < 0.001, transwell analysis ****P < 0.0001). This indicated that CoCl2-induced hypoxia promoted the proliferation and migration of HRECs while knockdown of MALAT1 inhibited the proliferation and migration of HRECs. Therefore, MALAT1 knockdown suppresses hypoxia-induced dysfunction in HRECs.
3.3 Knockdown of MALAT1 Inhibits Hypoxia-induced Neovascularization and Expression of Inflammatory Factors in HRECS
Next, to explore the regulatory role of MALAT1 in RNV/ROP formation, we investigated the mechanism of MALAT1 in hypoxia-induced angiogenesis. The results of detecting the angiogenic activity of HRECs by the tube formation assay showed that HRECs in the CoCl2 group exhibited a higher number of branches compared with the Control group (Fig. 3A, B, **P < 0.01). Compared with the CoCl2 + NC group, the network structure of the CoCl2 + RNAi group was significantly reduced (**P < 0.01), suggesting that MALAT1-RNAi intervention significantly reduced the angiogenic ability of HRECs. Western blotting results showed that, compared with the CoCl2 + NC group, the expression levels of VEGF, IL-1β, and TNF-α in the CoCl2 + RNAi group were significantly decreased (Fig. 3C, D, **P < 0.01). This suggests that MALAT1 knockdown can significantly reduce the inflammatory response in hypoxia-induced HRECs.
3.4 MALAT1 Regulates MMP-2 Expression by Sponging Mir-106a-5p MALAT1 mRNA, which Is Highly Expressed in the Retina of OIR Mice
We found that miR-106a-5p and MMP-2 were also differentially expressed. The qRT-PCR results showed that compared with the CoCl2 + NC group, the MMP-2 mRNA expression in the CoCl2 + RNAi group was significantly decreased (Fig. 4A, *P < 0.01). The western blotting results showed that compared with the CoCl2 + NC group, the expression of MMP-2 was lower in the CoCl2 + RNAi group (Fig. 4C, D, *P < 0.05). Similarly, overexpression of miR-106a-5p effectively reduced MMP-2 mRNA and protein levels (Fig. 4B, E, F, qRT-PCR**P < 0.01, western blotting *P < 0.05). The dual-luciferase reporter assays further verified the target-binding relationship. In the MALAT1-WT group, transfection of the miR-106a-5p mimic significantly reduced the relative activity of luciferase compared to the NC mimic (Fig. 5C, ****P < 0.0001), indicating that there was an interaction. Similarly, the results of the dual-luciferase reporter gene assay demonstrated a direct interaction between MMP-2 and miR-106a-5p (Fig. 5D, ****P < 0.0001).
Next, we examined the expression of miR-106a-5p in HRECs from the control, CoCl2, CoCl2 + NC, and CoCl2 + RNAi groups by qRT-PCR (Fig. 5E). As expected, in the CoCl2 group, the expression of miR-106a-5p was lower than that in the Control group (****P < 0.0001), while in the CoCl2 + RNAi group, miR-106a-5p expression was lower than that in the CoCl2 + NC group (****P < 0.0001). There was a corresponding change in miR-106a-5p levels when MALAT1 expression changed apparently. Therefore, we speculated that upregulation of MALAT1 expression in HRECs under hypoxia reduced the expression of has-miR-106a-5p, thereby promoting the expression of MMP-2. Taken together, MALAT1 can regulate the expression of MMP-2 by sponging miR-106a-5p, thereby affecting the biological function of HRECs.
3.5 Overexpression of miR-106a-5p Inhibits Hypoxia-induced Apoptosis and Migration of HRECs
MiR-106a-5p relative expression was determined using qRT-PCR analysis in the Control, CoCl2, CoCl2 + NC, and CoCl2 + mimics groups. The results showed that the expression of miR-106a-5p was lower in the CoCl2 group than the Control group (Fig. 6A, ****P < 0.0001). Compared to the CoCl2 + NC group, miR-106a-5p expression increased in the CoCl2 + mimics group (****P < 0.0001). The results of flow cytometry (Fig. 6D) showed that compared with the CoCl2 + NC group, the apoptosis rate of the CoCl2 + mimics group was significantly reduced (Fig. 6B, **P < 0.01). In addition, western blotting showed (Fig. 6B) that the CoCl2 + mimics group had decreased caspase-3 expression and increased Bcl-2 expression compared to the CoCl2 + NC group (Fig. 6C, *P < 0.05). The results of the wound healing and transwell experiments (Fig. 6E, H) showed that the mobility of the CoCl2 + mimics group was lower than that of the CoCl2 + NC group (Fig. 6G, I, wound-healing experiments: ***P < 0.001, migration experiments: ** P < 0.01). These results show that overexpression of miR-106a-5p can effectively inhibit cell apoptosis and migration.
3.6 Overexpression of miR-106a-5p Inhibits Angiogenesis and Expression of Inflammatory Factors in Hypoxia-induced HRECs
The results of the tube formation experiments showed that the CoCl2 + mimics group had less network structure than the CoCl2 + NC group (Fig. 7A, B, *P < 0.05). Western blotting showed that the expression levels of VEGF, IL-1β, and TNF-α in the CoCl2 + mimics group were lower than those in the CoCl2 + NC group (Fig. 7C, D, *P < 0.05, **P < 0.01).
3.7 Overexpression of miR-106a-5p Attenuates the Degree of Oxygen-induced Retinopathy
A retinal image of each group of 17-day-old mice is shown in Fig. 8D. In comparison with the control group, the avascular area of the retina in the OIR group was larger and showed more neovascularization. The non-perfusion area and neovascularization of the OIR-mimics group were significantly lower than those of the OIR-NC group (Fig. 8A–C, ****P < 0.0001). H&E staining showed that the retinal cells were neatly arranged in the Control group but disordered in the OIR group; moreover, the number of endothelial cell nuclei that broke through the inner limiting membrane and reached the vitreous cavity was higher in OIR group than that the Control group (Fig. 8D, E, ****P < 0.0001). In the OIR-mimics group, endothelial cells were neatly arranged and fewer nuclei broke through the inner limiting membrane than in the OIR-NC group (****P < 0.0001). It appears that upregulation of miR-106a-5p can reduce retinal lesions in HRECs overexpressing miR-106a-5p, consistent with our findings in HRECs overexpressing miR-106a-5p.
3.8 Overexpression of miR-106a-5p Suppresses Apoptosis and Inflammation in Retinal Tissue in Oxygen-induced Retinopathy
Compared with the OIR-NC group, the expression of MALAT1 was significantly decreased in the OIR-mimics group (Fig. 9A, ****P < 0.0001), and miR-106a-5p was significantly increased by qRT-PCR in the OIR model (Fig. 9B, ****P < 0.0001). Compared with the OIR-NC group, IL-1β, TNF-α and caspase-3 were downregulated in the OIR-mimics group (Fig. 9C–F, *P < 0.05, **P < 0.01, ****P < 0.0001), and Bcl-2 was upregulated (Fig. 9C, D, ****P < 0.0001). The immunohistochemistry results showed the same trend (Fig. 9G, H, ****P < 0.0001).
3.9 Overexpression of miR-106a-5p Inhibits MMP-2, Hypoxia-inducible Factor 1-α (HIF-1α) and Vascular Endothelial Growth Factor (VEGF) Expression
A higher level of MMP-2 mRNA expression was observed in the OIR-mimics group than in the miR-106a-5p group based on qRT-PCR results (Fig. 10A, ***P < 0.001). The western blot results showed that compared with the Control group, the expression of MMP-2, VEGF, and HIF-α was significantly increased in the OIR group, while in the OIR + mimics group, it was significantly lower than in the OIR + NC group (Fig. 10B, C, * P < 0.05, **P < 0.01, ***P < 0.001).Immunohistochemistry showed that the expression levels of the inflammatory factors IL-1β and TNF-α in the retina of the OIR group were significantly higher than those of the Control group, while the opposite trend was observed in the OIR + mimics group (Fig. 10D, E, ****P < 0.0001). This indicated that the overexpression of miR-106a-5p attenuated retinal inflammatory responses and suppressed the expression of VEGF and HIF-1α. VEGF and HIF-1α are two key factors that promote angiogenesis [12], and reducing their expression can effectively reduce angiogenesis, thereby controlling the occurrence and development of ROP.
ROP is a unique vascular proliferative retinal disease in premature infants, and the core of the disease is hypoxic damage. When retinal tissue is ischemic and hypoxic, the oxidative and antioxidant systems in the body are unbalanced and peroxides cannot be removed quickly. Excessive accumulation of reactive oxygen species (ROS) leads to endothelial cell damage and oxidative stress and promotes angiogenesis[16]. Angiogenesis is considered an important pathological process in ROP. Retinal neovascularization results in numerous anatomical and physiological changes that ultimately lead to vision loss[17]. In recent years, many lncRNAs have been shown to be closely associated with the occurrence and development of retinopathy. For example, the lncRNA MALAT1 is sensitive to variations in oxygen levels, ischemia and hypoxia can cause endothelial cell damage by inducing it high expression. It has been demonstrated that MALAT1 reduces apoptosis in brain microvascular endothelial cells (ECs) after ischemia[18], facilitates the tube formation of human umbilical vein endothelial cells and the function of their endothelial barriers [19], and stimulate the proliferation of HRECs induced by high glucose; moreover, inhibiting MALAT1 can induce the phenotype of endothelial cells to switch to migration, inhibit proliferation, and reduce retinal neovascularization[20, 21]. Similarly, microRNAs (miRNAs) have been reported to be involved in the physiological and pathophysiological processes of retinopathy. Studies have shown that MALAT1 can promote the angiogenesis and proliferation of brain microvascular endothelial cells cultured under hypoxia by targeting miR-145[22]. In addition, Liu et al.[23] found that MALAT1 can promote neovascularization in diabetic retinopathy by regulating the miR-125b/VE-cadherin (vascular endothelial cadherin, VE-cadherin) axis while MALAT1 deletion can inhibit the neovascularization expression of related proteins (VEGF, VE-cadherin, β-catenin, matrix metalloproteinase 2, matrix metalloproteinase 9, etc.).
Our previous study of the OIR model revealed significantly higher expression of MALAT1 in retinal tissue and knockout of MALAT1 in vivo could significantly reduce the degree of retinopathy and inflammatory response, thereby effectively inhibiting RNV[24]. On this basis, we predicted that miR-106a-5p may be involved in the regulation of MMP-2 expression through MALAT1. In in vivo experiments, an OIR model was employed that simulates the relative hypoxic phases of ROP induced by hyperoxia and normoxia[25, 26]. The experimental results showed that the OIR model had a significant decrease in the expression of miR-106a-5p and an increase in the expression of MALAT1 and MMP-2; in addition, overexpression of miR-106a-5p could reverse these effects. We also found that in the OIR mouse model, the expression of apoptotic and inflammatory factors increased while the expression of anti-apoptotic factors decreased. Retinal apoptosis and inflammatory responses were reduced after the in vivo injection of miR-106a-5p mimics. In addition, due to retinal ischemia and hypoxia, the stimulation of important retinal neovascular disease regulators, such as VEGF and HIF-1α, increases and promotes endothelial cell apoptosis and migration, leading to RNV formation[27]. Wu et al.[28]found that HIF-1α and VEGF-A are involved in hypoxia-induced retinal angiogenesis. After overexpression of miR-106a-5p, we found that the expression of HIF-1α and VEGF decreased in OIR mice, indicating that overexpression of miR-106a-5p had an antagonistic effect on RNV formation.
In in vitro experiments, CoCl2 was used to induce hypoxia in HRECs to mimic the relative hypoxic state of OIR[29]. We found that MALAT1 knockdown inhibited the hypoxia-induced proliferation and migration of HRECs. Furthermore, we demonstrated that MALAT1/miR-106a-5p/MMP-2 is involved in hypoxia-induced angiogenesis and inflammatory responses in HRECs. After the knockdown of MALAT1 and overexpression of miR-106a-5p, the mRNA and protein levels of MMP-2 were significantly inhibited, followed by decreased angiogenesis and VEGF and inflammatory mediator levels. These results suggest that under hypoxic conditions, downregulation of MALAT1 reduces the proliferation, migration, angiogenesis, and inflammatory response of HRECs and protects the retina by enhancing the expression of miR-106a-5p and inhibiting the expression of MMP-2. MMP-2 is an important member of the matrix metalloproteinase (MMP) family. MMP mainly participates in the formation of pathological NV by hydrolyzing extracellular matrix (ECM) proteins and plays an important role in several types of retinopathies. Within the MMP family, MMP-2 has been reported to be selectively associated with neovascular processes in retinopathy such as ROP and DR[30]. By upregulating miR-106a-5p and reducing the level of MMP-2, we inhibited the apoptosis of HRECs and the expression of inflammatory factors, which significantly reversed hypoxia-induced vascular endothelial cell dysfunction and slowed the progression of RNV/ROP. Therefore, we demonstrated a novel function for MALAT1/miR-106a-5p/MMP-2 in ROP progression.
There remain many shortcomings in the current treatment methods for RNV, such as surgery, laser therapy, and intravitreal injection of anti-VEGF drugs. Therefore, new therapeutic targets must be identified. The current research hotspot is gene therapy mediated by shRNA, which has been shown to be effective in reducing the number of treatments and sustaining the effect[31–35]. Combined with our findings, the inhibition of MALAT1/miR-106a-5p/MMP-2 can effectively antagonize RNV formation in ROP. Interventions targeting this pathway may be an alternative approach for the treatment of ROP and other proliferative retinopathies, and we look forward to extensively exploring such approaches to elucidate the impact and therapeutic potential of MALAT1 on the pathological process of ROP and achieve promising clinical applications.
Data Availability Statement
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author.
Conflict of Interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Author Contributions
ZjL: Creating the original draft—conceptualization, methodology, formal analysis, and writing. KyL and SyM: analyzed the data. ZbW: resources and validation. YD: funding acquisition and supervision. Authors all contributed to the article and approved the final version.
Funding
The study was funded by Liaoning province's Department of Education (QNZR2020010) and Shengjing Hospital of China Medical University's 345 Talent Project (40B).
Acknowledgments
For the research environment provided by Shengjing Hospital Affiliated to China Medical University and guidance provided by tutor YD regarding experimental ideas and discussions, we are grateful. We also thank the co-authors for their help.
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No competing interests reported.