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LncRNA TUG1 Promotes Apoptosis, Invasion, and Angiogenesis of Retinal Endothelial Cells in Retinopathy of Prematurity via MiR-145-5p

Purpose Retinopathy of prematurity (ROP) is a common retinal vascular disease in premature neonates. In recent years, there is increasing evidence that the long non-coding RNA taurine upregulated gene 1 (TUG1) plays a regulatory role in vascular diseases, suggesting a potential role for TUG1 in vascular endothelial cells. We hypothesized that TUG1 may be associated with ROP. Our aim, therefore, was to explore the biological functions of TUG1 in aberrant retinal development. Methods We used the mouse oxygen-induced retinopathy (OIR) model to simulate the pathological changes of retinal in ROP. Quantitative real-time polymerase chain reaction was used to detect the expression of TUG1, miR-145-5p and cellular communication network factor 1 (CCN1). Human retinal endothelial cells (HRECs) were treated with CoCl2 to mimic hypoxia conditions. Cellular functional changes were observed after transfection with RNA interference (RNAi)-TUG1 and miR-145-5p mimics. The apoptosis of HRECs was detected by flow cytometry, the migration ability was detected by wound healing and transwell migration assays, and the ability of angiogenesis was detected by tube formation assay. The potential binding sites between TUG1, miR-145-5p, and CCN1 were verified by dual-luciferase reporter assays. The degree of retinopathy was evaluated by staining retinal sections with hematoxylin and eosin, and the expression of CCN1, HIF-1α, VEGF, caspase-3, Bcl-2, IL-1β, and TNF-α protein was analyzed by Western blotting and immunohistochemistry. Results In the retina tissue of OIR mice, TUG1, miR-145-5p, and CCN1 were differentially expressed. Knocking down TUG1 attenuated apoptosis, migration, and angiogenesis induced by hypoxia on HRECs, as did miR-145-5p overexpression. Results from reporter assays indicate direct interactions between TUG1, miR-145-5p, and CCN1. Intravitreal injection of miR-145-5p mimics reduced the degree of retinopathy. Conclusion TUG1 acts as a molecular sponge of miR-145-5p to regulate CCN1 expression and thus regulate the development of retinal neovascularization. This regulatory mechanism may provide a new theoretical basis for the prevention and treatment of ROP.


INTRODUCTION
Retinopathy of prematurity (ROP) is a retinal vascular disease that occurs in preterm and low birth weight infants. While the details of the pathogenic process are not clear, it is believed that the formation of retinal non-perfusion areas and pathological retinal neovascularization (RNV) lead to ROP (1,2). At present, retinal laser photocoagulation and vitreous injection of antivascular endothelial growth factor (VEGF) drugs are commonly used to control the development of ROP. However, laser photocoagulation may damage the retina, and drugs injected into the vitreous cavity may enter the blood circulation, adversely affecting the underdeveloped organ systems of premature infants. Even if drugs are combined with laser therapy, there are serious adverse reactions (3)(4)(5). Therefore, at present, anti-VEGF drugs are still used cautiously in the treatment of ROP. In light of these drawbacks, the prevention and treatment of ROP is still an unsolved problem, and there is an urgent need to identify new strategies to combat this disease.
Long non-coding RNAs (lncRNAs) are a class of RNA transcripts that are greater than 200 bp in length and have almost no potential to encode proteins (6). The lncRNA taurine upregulated gene 1 (TUG1) was discovered relatively early in the history of lncRNA investigations, and it is indispensable in the development of the retina (7). It has also been implicated in various disease-linked cellular processes, including cell proliferation, cell differentiation, cell invasion, radiation resistance, angiogenesis, and inflammation (8,9). In particular, it plays an important role in neovascularization and the malignant degree and prognosis of many tumor types (10). It plays an important role in biological regulation by acting as a competitive endogenous RNA (or sponge) for microRNAs (miRNAs), thereby regulating downstream mRNAs (11). The miRNAs that bind to TUG1 include miR-1299, miR-186-5p, miR-212-3p, miR-145, miR-26a, and so on (12)(13)(14). MiRNAs are endogenous noncoding small RNAs that are 21-23 nucleotides in length and highly conserved in evolution (15,16). LncRNAs can compete with miRNAs to regulate the proliferation and migration of human retinal endothelial cells (HRECs) and the formation of RNV in diabetic retinopathy (17). Based on the fact that the molecular mechanism of RNV in ROP is similar to that of diabetic retinopathy, we speculated that lncRNAs and miRNAs may also be involved in the regulation of ROP. Cellular communication network factor 1 (CCN1) is a stromal cell protein involved in vascular development and tissue repair. In our previous study, we found that CCN1 is highly expressed during RNV in the formation of ROP (18), and a study that analyzed human clinical specimens also showed that the level of CCN1 increased in some ocular vascular complications (19). Results from bioinformatics analyses (via the starBase database 2.0. 1 ) (20) predict that miR-145-5p is a target of TUG1 and CCN1 and suggest that TUG1 may affect the expression of CCN1 by regulating miR-145-5p. Therefore, this study aimed to investigate the expression and function of the lncRNA TUG1 and miR-145-5p in a mouse model of oxygen-induced retinopathy (OIR) and HRECs. Our results provide new insights for clinical investigations focused on improving treatments for ROP. Mice were reared in the standard pathogen-free facility for experimental animals at the Benxi base of Shengjing Hospital under temperature conditions of 23 ± 2 • C, with a 12-h light/dark cycle. The pups were randomly divided into the control, OIR, OIR-negative control (NC), and OIR-mimics groups, with 60 pups per group. An OIR model was established using the classical Smith method (21). The pups in the control groups were placed in normoxia up to postnatal day (PD) 17. On PD 7, pups in OIR, OIR-NC, OIR-mimics groups were placed in a hyperoxia chamber (75 ± 2%) for 5 days and subsequently removed to normoxic condition for a further 5 days up to PD 17. OIR-NC and OIR-mimics groups received intravitreal injection under isoflurane inhalation anesthesia on PD12. The pups in OIR-NC group received intravitreal injection of 0.5 µL of miR-145-5p mimics negative control reagent (miR-145-5p NC mimics) + 0.5 µL of animal infection reagent (IVG2101) (InvivoGene, China) + 0.5 µL of PBS, and the OIR-mimics group was treated with 0.5 µL of miR-145-5p mimics reagent (miR-145-5p mimics) + 0.5 µL of animal infection reagent + 0.5 µL of PBS. The sequences of miR-145-5p NC mimics were as follows: sense: 5 -UUCUCCGAACGUGUCACGUTT-3 ;

RNA Extraction and Quantitative Real-Time Polymerase Chain Reaction
Total RNA was extracted from cultured cells and retina tissue using TRIzol (Takara Biotechnology, Otsu, Japan), and the expression of TUG1, miRNA-145-5p, and CCN1 mRNA was detected by qRT-PCR. cDNA was synthesized using the Mir-X miRNA First-Strand Synthesis kit (Cat. No. 638313, Takara Biotechnology, Otsu, Japan) and the PrimeScript RT reagent kit with gDNA Eraser (Code No. RR047Q, Takara Biotechnology). PCR was then performed on a 7500 Fast Real-Time PCR machine (Applied Biosystems, Warrington, United Kingdom) using TB Green R Premix Ex Taq TM II (Tli RNaseH Plus, code no. RR820A, Takara Biotechnology). The reaction conditions were as follows: 40 cycles of denaturation for 30 s at 95 • C, amplification for 3 s at 95 • C and 30 s at 60 • C. β-Actin and U6 were used as internal reference genes for mRNA and miRNA analyses, respectively. For quantitative analysis of differential expression, data were processed using the 2 − Ct method, with the mRNA expression presented as the relative fold change compared to β-actin, and miRNA expression presented as the relative fold change compared to U6. The primers (Shenggong, Shanghai, China) used are shown in Table 1.

Flow Cytometry for Apoptosis
Twenty-four hours after transfection, HRECs were treated with 200 µmol/L CoCl 2 for 24 h. Then, the cells were collected by treating with trypsin without EDTA and subjected to AnnexinV-PE/7-AAD labeling using an apoptosis detection kit (Vazyme, Nanjing, China). The cells were incubated at room temperature and protected from light for 10 min according to the manufacturer's instructions and analyzed by flow cytometry (BD, FACSCalibur, NJ, United States). Cell Quest Pro software (Becton-Dickinson, CA, United States) was used for data analysis.

Wound Healing Assay
Human retinal endothelial cells were uniformly seeded into a sixwell culture plate, and three horizontal lines were marked on the bottom of each well. The cells were transfected and treated with CoCl 2 as described earlier. When the cells reached a confluency of approximately 90%, the tip of a 10 µL micropipette was used to make a uniform scratch through the cell monolayer, perpendicular to the horizontal line. The disrupted cell mass was gently washed off with PBS, and the remaining adherent cells were incubated in a serum-free culture medium. After 48 h, wound healing was examined and photographed.

Transwell Migration Assay
The cells were transfected and treated with CoCl 2 as described earlier. Next, 3 × 10 4 cells were suspended in 200 µL of serum-free RPMI1640 medium and transferred to the upper chamber of the transwell insert in a 24-well plate (Corning, NY, United States), and 500 µL of medium containing 10% FBS was added to the lower chamber. The cells were incubated at 37 • C for 24 h, after which the remaining cells in the upper chamber were gently scraped off with cotton swabs. The transwell membrane was then fixed with 4% paraformaldehyde for 20 min, washed with PBS, stained with 0.5% hematoxylin solution for 20 min, and washed again with PBS. Finally, we analyzed the number of cells on the bottom of the membrane using an inverted microscope (ECLIPSE Ni-U, Nikon, Tokyo, Japan). For each well, the average number of cells was calculated from five randomly selected fields of view.

Tube Formation Assay
The bottom of a 96-well plate was evenly coated with 50 µL of Matrigel (Corning, NY, United States) diluted 2:1 in a basic medium and incubated at 37 • C for 30 min to form a gel. The cells were suspended in a medium containing 10% FBS, and 1 × 10 4 cells were seeded per well in the Matrigel-coated 96well plate. After 6 h of incubation, each well was examined and photographed using an inverted microscope (ECLIPSE Ni-U, Nikon). Each treatment group comprised three wells, and 1-2 fields of view were captured for each well. The overall experiment was repeated five times. In each field of view, we counted the number of nodes and meshes and measured the total branching length of the tubes.

Dual-Luciferase Reporter Assays
Dual luciferase reporter assays were used to verify binding of miR-145-5p with TUG1 and CCN1. The sites in miR-145-5p that facilitate binding with TUG1 and CCN1 were predicted via the starBase database. An expression vector was designed and constructed. First, 5 × 10 3 HEK293T cells were seeded into each well in a 96-well plate. After 12 h, 3 pmol miR-145-5p mimics or miR-145-5p NC mimics were co-transfected with 0.1 µg TUG1-WT or TUG1-MUT using Lipofectamine 3000, according to the manufacturer's instructions. After a further 36 h, the cells were lysed with 1 × passive lysis buffer from the Dual-Luciferase Reporter Assay System (Promega, WI, United States), and luciferase activity was measured on a luminometer (Synergy H1 microplate reader, BioTek, VT, United States). Firefly luciferase activity was normalized to Renilla luciferase activity. Each transfection reaction was performed in triplicate. The same procedure was used for the co-transfection of miR-145-5p mimics or miR-145-5p NC mimics with CCN1-WT or CCN1-MUT.

Retinal Vascular Staining
Under isoflurane inhalation anesthesia, eyeballs from 17-dayold mice were removed and fixed in 4% paraformaldehyde overnight. The limbus cornea was incised and the anterior segment and vitreous body were removed. Retinal tissue was separated and cleaned with PBS, the Isolectin IB4 Alexa Fluor R dye conjugate (Invitrogen, United States) was dissolved in PBS solution containing 1% TritonX-100 (Solarbio, Beijing, China), and incubated with the retinal tissue at 4 • C overnight. The retinal tissue was taken out the next day, cleaned with PBS, placed on glass slides, and cut into four sections under the microscope. The slices were sealed with an anti-quenching agent and photographed with a fluorescence microscope (ECLIPSE Ni-U, Nikon). ImageJ software 1.51 (NIH, United States) was used to calculate the retinal avascular area.
Detailed steps are as follows: 1. Open the picture, click Image → Type → 8-bit.

Hematoxylin and Eosin Staining
The eyeballs of 17-day-old mice were fixed in 4% paraformaldehyde overnight, embedded in paraffin, and cut into 4 µm thick sections. The slices were dried at 60 • C for 6, dewaxed, stained with hematoxylin and eosin (HE), dehydrated, and sealed with neutral gum. The number of nuclei penetrating the inner limiting membrane of the retina was directly counted by the double-blind method.

Western Blotting
The treated cells and retinal tissue were lysed in RIPA lysis buffer (Solarbio) containing 1% PMSF (Solarbio), and the protein concentration was determined using BCA assay (BCA Protein Quantification Kit, Vazyme, Nanjing, China). Then, protein samples were separated with a 10% fast electrophoresis gel preparation kit (EpiZyme, Shanghai, China) and transferred to a PVDF membrane (Merck Millipore, Burlington, MA, United States). After blocking with 5% bovine serum albumin for 1 h, membranes were incubated in primary antibody overnight at 4 • C. The primary antibodies used were rabbitderived anti-CCN1 (1:1,000) and rabbit-derived anti-tubulin (1:1,000) (both Immunoway). The secondary antibody was horseradish peroxidase-labeled goat anti-rabbit IgG (H + L)

Statistical Analysis
All analyses were performed in GraphPad Prism 8.0 software (GraphPad Software Inc., CA, United States). All values are presented as the mean ± standard deviation (SD) of three or more independent experiments. Statistical analysis was performed using Student's t-test or one-way ANOVA. * P < 0.05, * * P < 0.01, and * * * P < 0.001 indicate significant differences.

Tug1 Expression Was Upregulated and MiR-145-5p Was Down-Regulated in Oxygen-Induced Retinopathy Mice
We analyzed the expression of Tug1 and miR-145-5p in the control group and the OIR group by using qRT-PCR. The results showed that the expression of Tug1 in the OIR group was significantly higher than that in the control group (Figure 1A, * * * P < 0.001), while the expression of miR-145-5p significantly lower ( Figure 1B, * * P < 0.01).

Knocking Down Taurine Upregulated Gene 1 Inhibits Apoptosis, Migration, and Angiogenesis in Human Retinal Endothelial Cells Induced by Hypoxia
As shown in Figure 2A, TUG1 expression was successfully knocked down in normal HRECs when transfected with TUG1-RNAi, and TUG1-RNAi 1 was selected for subsequent experiments. Next, we determined the relative expression of TUG1 in the control, CoCl 2 , CoCl 2 + NC, and CoCl 2 + RNAi groups ( Figure 2B). TUG1 expression was significantly higher in the CoCl 2 group compared to the control ( * * * P < 0.001), and expression of the TUG1 was significantly lower in the CoCl 2 + RNAi group compared to the CoCl 2 + NC ( * * * P < 0.001). These results suggest that TUG1-RNAi successfully inhibited the increase in TUG1 induced by hypoxia. Flow cytometry analysis indicated that the apoptosis was increased in the CoCl 2 group compared with the control (Figures 2C,D, * * * P < 0.001) and was significantly inhibited in the CoCl 2 + RNAi group compared with the CoCl 2 + NC ( * * * P < 0.001). The wound healing and transwell migration assays showed that treatment with CoCl 2 promoted HREC migration compared with that in the control (Figures 2E-H, wound healing assay * * * P < 0.001, transwell assay * * * P < 0.001), the migratory ability of HRECs in the CoCl 2 + RNAi group was lower than that in the CoCl 2 + NC group (wound healing assay * * * P < 0.001, transwell migration assay * * * P < 0.001). Together, these data indicate that hypoxia induced by CoCl 2 promoted HREC migration and knocking down TUG1 inhibited the migratory ability of these cells. The formation of RNV is related to the tube-forming ability of endothelial cells, and therefore, we examined the angiogenic activity of HRECs using a tube formation assay. The results demonstrated that, compared with the control group (Figures 2I,J), the CoCl 2 group exhibited a greater number of meshes ( * * * P < 0.001), which promoted tube formation in the HRECs. Compared with the CoCl 2 + NC group, the CoCl 2 + RNAi group exhibited fewer meshes ( * * * P < 0.001), indicating that loss of TUG1 reduces the angiogenic capacity of HRECs.

MiR-145-5p Interacts With Taurine Upregulated Gene 1 and Cellular Communication Network Factor 1
Bioinformatic analyses using the starBase interface indicated potential binding sites between TUG1 and miR-145-5p ( Figure 3A). The transfection efficiency of miR-145-5p mimics was confirmed by qRT-PCR ( Figure 3B, * * * P < 0.001). Results from luciferase reporter assays showed that in the TUG1-WT group, transfection with miR-145-5p mimics significantly reduced relative luciferase activity compared with that of the NC mimics ( Figure 3C, * * * P < 0.001), suggesting an interaction between them. Our bioinformatic analyses also predicted the presence of potential binding sites between miR-145-5p and CCN1 (Figure 3A). The results of luciferase reporter assays also indicated a direct binding interaction between CCN1 and miR-145-5p (Figure 3D, * * * P < 0.001). We next examined the expression of miR-145-5p in HRECs in the control, CoCl 2 , CoCl 2 + NC, and CoCl 2 + RNAi groups using qRT-PCR ( Figure 3E). As expected, the expression of miR-145-5p in the CoCl 2 group was lower than that in the control group ( * * * P < 0.001), while miR-145-5p expression was higher in the CoCl 2 + RNAi compared to that in the CoCl 2 + NC group ( * * * P < 0.001). These results suggest that changes in TUG1 expression can produce corresponding changes in miR-145-5p levels.

Overexpressing MiR-145-5p Inhibits Apoptosis, Migration, and Angiogenesis in Human Retinal Endothelial Cells Induced by Hypoxia
We further explored the influence of miR-145-5p on the biological functions of HRECs. The relative expression of miR-145-5p in the control, CoCl 2 , CoCl 2 + NC, and CoCl 2 + mimics groups was determined using qRT-PCR analysis ( Figure 4A).
The results showed that miR-145-5p expression decreased in the CoCl 2 group compared to the control group ( * P < 0.05) but increased in the CoCl 2 + mimics group compared to the CoCl 2 + NC group ( * * * P < 0.001). The percentage of apoptotic cells was reduced in the CoCl 2 + mimics group compared with the CoCl 2 + NC group (Figures 4B,C, * * * P < 0.001), suggesting that the overexpression of miR-145-5p inhibits apoptosis. The results from the wound healing and transwell migration assays revealed that the migration rate of the CoCl 2 + mimics group was lower than that of the CoCl 2 + NC group (Figures 4D-G, wound healing assay * * * P < 0.001, transwell migration assay * * * P < 0.001), supporting the role of overexpression of miR-145-5p in inhibiting cell migration. The results from the tube formation assay revealed a similar trend (Figures 4H,I). Compared with the CoCl 2 + NC group, the CoCl 2 + mimics group exhibited fewer meshes ( * * * P < 0.001).

Overexpression of MiR-145-5p Can Reduce the Degree of Oxygen-Induced Retinopathy Retinal Lesions
Retinal images of 17-day-old mice are shown in Figure 6A. Compared with the control group, the OIR group had a larger non-perfusion area and more neovascularization, while the OIR-mimics group injected with miR-145-5p mimics had a significantly lower non-perfusion area and neovascularization than the OIR-NC group (Figures 6A-C, * * * P < 0.001).
Hematoxylin and eosin staining revealed that the retinal cells in the control group were arranged neatly, while those in the OIR group were disorderly, and the number of endothelial nuclei breaking through the inner limiting membrane into the vitreous lumen was significantly increased (Figures 6D,E, * * * P < 0.001). Compared with the OIR-NC group, the endothelial cells in the OIR-mimics group were arranged neatly and the number of endothelial nuclei breaking through the inner boundary membrane decreased significantly ( * * * P < 0.001). These results are consistent with our observations of HREC overexpressing miR-145-5p that exhibited reduced migration and angiogenesis capacity. From outside to inside, the retina is as follows: pigment epithelium, optic rod cone layer, outer limiting membrane, outer nuclear layer, outer reticular layer, inner nuclear layer, inner reticular layer, ganglion cell layer, nerve fiber layer, and inner limiting membrane. Further inside the inner limiting membrane is the vitreous body.
Over Expression of MiR-145-5p Inhibits the Expression of Cellular Communication Network Factor 1, Hypoxia-Inducible Factor-1 α, and Vascular Endothelial Growth Factor The qRT-PCR results showed that CCN1 mRNA expression was increased in OIR and miR-145-5p decreased in the OIR-mimics group ( Figure 8A, * * * P < 0.001). Western blot results showed that the expression of CCN1, HIF-α, and VEGF were significantly increased in the OIR group compared with the control group, while in the OIR-mimics group the same were significantly decreased compared with the OIR-NC group (Figures 8B,C,E,F, * * * P < 0.001). Immunohistochemistry also showed the same trend (Figures 8D,G, * * * P < 0.001).

DISCUSSION
The increase in the mean age of mothers and global advances in perinatal medicine have resulted in a high number of premature births and survival, leading to an increased rate of incidence of ROP. The development of ROP is generally believed to be related to the relative hypoxia of the retina. When premature neonates are exposed to supplemental oxygen in incubation chambers, returning them to a normoxic environment can cause vasoconstriction and occlusion, which is mainly seen in the developing retinal vessels (23). Exposure to high levels of oxygen may lead to an increase in potentially destructive reactive oxygen species (ROS), and the return to normal oxygen environment reduces antioxidants to neutralize ROS in the retina of preterm infants, leading to endothelial cell damage, apoptosis, inflammation, and capillary constriction (24,25).
The OIR model is commonly used to study retinal vascular diseases, including ROP because it simulates the relative hypoxia stage of ROP created by hyperoxia followed by normoxia (26,27). Many previous reports have described miR-145-5p as a target gene of TUG1, and that knockdown of TUG1 can inhibit cell migration and damage, which may have a protective effect on cells via targeting miR-145-5p (28)(29)(30). In our study, we found that the expression of the lncRNA Tug1 was upregulated and the expression of miR-145-5p was down-regulated in the OIR mouse model. In our in vitro experiments, we induced hypoxia in HREC with CoCl 2 to simulate the relative hypoxia state of OIR, and this is method has been used previously by Du et al. to study  OIR (22). Our results also showed that when TUG1 was knocked down or miR-145-5p was overexpressed, apoptosis, migration, and angiogenesis were inhibited, which have a protective effect on cells. Furthermore, the expression of CCN1 mRNA and protein levels were both affected, suggesting that CCN1 is regulated prior to CCN1 mRNA translation. Therefore, we propose that TUG1 acts as a molecular sponge of miR-145-5p to regulate CCN1 expression, the dual-luciferase reporter assays also confirmed the binding relationship among them.
The changes in inflammatory and apoptotic factors are observed in many retinal diseases, including ROP (27,31). We also found the expression of apoptotic and inflammatory factors increased and anti-apoptotic factors decreased in OIR mouse models. When we overexpressed miR-145-5p in vivo, retinal apoptosis and inflammation were reduced. Furthermore, it is generally believed that hypoxia leads to the increase of hypoxiainducible factor-1 α (HIF-1α) and VEGF, which leads to the apoptosis and migration of endothelial cells resulting in RNV (32). In our study, HIF-1α and VEGF were also significantly altered in OIR mice, and overexpressing of miR-145-5p in vivo, decreased retinal apoptosis and neovascularization significantly. These results indicate that miR-145-5p overexpression has a protective effect on RNV/OIR. Moreover, we have previously demonstrated that knocking down TUG1 can reduce retinal apoptosis, inflammation, and damage in OIR mice (33). In conclusion, TUG1 acts as a molecular sponge of miR-145-5p to regulate CCN1 expression and TUG1/miR-145-5p/CCN1 together are involved in the occurrence and development of RNV in OIR mice.
The current clinical treatment for ROP is mainly vitreous injection. However, the effects of vitreous injections are shortlived and most patients may require multiple injections to achieve the effect of treatment, which increases the risk of infection. Therefore, it is essential to study alternative target molecules. One of the advantages of gene therapy is that it can shorten the treatment time and work continuously (34,35). Combined with our previous and current studies, it can be seen that a single knockdown of TUG1 or overexpression of miR-145-5p in vivo can achieve good therapeutic effects. Our findings reveal gene therapy as a potential long-term treatment for ROP and provide some new insights for gene therapy of neovascularization diseases. However, gene regulation is complex, and gene therapy technology as clinical treatment is not very mature, therefore, the safety of injection into the eye and the feasibility of gene therapy as a clinical application needs further research. However, we believe that with more research, gene therapy will solve not only ROP but also many other medical conditions.

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.

ETHICS STATEMENT
The animal study was reviewed and approved by the Medical Ethics Committee of Shengjing Hospital of China Medical University. This committee approved the use of animals for the experiments (ethics code: 2018PS239K) on 1 March 2018.

AUTHOR CONTRIBUTIONS
YxW: conceptualization, methodology, formal analysis, and writing-original draft preparation. YeW: data curation and visualization. XW: investigation and software. YM: resources and validation. ZL: writing-reviewing and editing. YD: funding acquisition and supervision. All authors contributed to the article and approved the submitted version.