MicroRNA-421 protects against chronic intermittent hypoxia-induced vascular endothelial cell injury by targeting TLR4

Purpose: To investigate the role of miR-421 in endothelial cell injury caused by chronic intermittent hypoxia (CIH), and to unravel the mechanism of action. Methods: A rat aortic endothelial cell model of CIH was established by 18-h exposure to hypoxic treatment. Cell viability was evaluated by MTT while cell apoptosis was determined by flow cytometry. Cellular reactive oxygen species (ROS) levels were assessed by cellular reactive oxygen species (ROS) assay kit. The mRNA and protein levels were also determined. Sprague Dawley rats were used to establish a rat CIH model over a 6-week hypoxic exposure. The degree of lung and renal injuries in the rats were observed by HE staining. Results: MiR-421 was downregulated and toll-like receptor 4 (TLR4) upregulated in CIH cells compared to control cells. Treatment of CIH cells decreased their viability, increased cellular ROS levels, promoted cell apoptosis, and caused changes in protein levels of apoptosisand inflammation-related genes. However, miR-421 mimics reversed these results caused by CIH treatment. Dual luciferase reporter assay verified that TLR4 was targeted by miR-421. Moreover, TLR4 overexpression suppressed the protective effect of miR-421 on CIH cells. Finally, miR-421 agomiR inhibited CIH-induced TLR4 upregulation in rats. Histopathological examinations confirmed that miR-421 agomiR inhibited CIHinduced injury and collagen deposition in rat lungs and kidneys. Conclusion: MiR-421 protects vascular endothelial cells against CIH-induced injury in rats by targeting TLR4, which may provide a biomarker for the diagnosis and treatment of CIH-induced injury.


INTRODUCTION
Obstructive sleep apnea syndrome (OSAS) is a common condition resulted from recurrent episodes of complete or partial obstruction in upper airway [1]. It may be caused by obesity, advanced age, male gender, alcohol abuse, and nasal obstruction [2]. It is well-known that OSAS can cause damage to multiple organs and systems, including the cardiovascular system, nervous system, lung, and kidney, [3][4][5]. Importantly, chronic intermittent hypoxia (CIH) is a characteristic of OSAS [4] and a key factor of oxidative stress that induce the release of reactive oxygen species (ROS), which subsequently results in systemic inflammation [1]. Previous studies confirmed that CIH is responsible for the dysfunction and injury of endothelial cell [6,7].
However, the relationship between miR-421 and CIH-induced endothelial cell damage is unclear. Besides, previous reports demonstrated that TLR4 had important roles in the pathogenesis of CIH-induced injury [19,20]. Thus, to determine whether miR-421 protects endothelial cells against CIH-induced damage via targeting TLR4, a series of in vivo and in vitro experiments were performed.

Animal model
All experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals of the Institutional Animal Care and Use Committees (IACUCs) of the United States [21] and approved by the institutional Animal Ethical and Welfare Committee (approval no. 2019-802). Forty-five Sprague Dawley rats (male, 200±20 g; 6-week-old; from the Laboratory Animal Research Center of Fourth Military Medical University) were grouped as follows: sham, CIH+NC agomiR, and CIH+miR-421 agomiR (n=15 in each group). Rats in the CIH+NC agomiR and CIH+miR-421 agomiR groups were kept in the chamber (Oxycycler model A84; BioSpherix) for 30 cycles/h for 8 h daily (from 9 am to 5 pm) for 6 weeks. In each 2-min cycle, oxygen concentration was dropped to 8% for 30sec, maintained for 50sec, and then increased to 20% for 40sec. Sham rats were placed in the chamber with 20% oxygen throughout the experiment. Besides, rats in the CIH+NC agomiR and CIH+miR-421 agomiR groups were injected through the tail vein with NC agomiR or miR-421 agomiR at a concentration of 15 mg/kg/day twice a week. At week 6, blood was collected through cardiac puncture before euthanization. Lungs and kidneys were collected for further analysis.

Flow cytometry analysis
Cells from different groups were washed with phosphate-buffered saline and then stained (Annexin V-fluorescein isothiocyanate and propidium iodide) for 10 min. Apoptotic cells were examined using a flow cytometer (Beckman Coulter, Brea, CA, USA).

Quantitative polymerase chain reaction (qPCR)
The RNA from cells was extracted and cDNA was synthesized. qPCR was conducted with SYBR Green quantitative RT-PCR kits (Sigma). Relative expression levels were calculated as ratios normalized against those of GAPDH, using the following primer sequences:

Histopathological examination
Lungs and kidney tissue were fixed (10% formalin) and embedded(paraffin). Hematoxylineosin (HE) or Masson staining was performed on 3-μm serial sections. All sections were observed under an optical microscope.

Statistical analysis
Data are presented as mean ± SD, and each experiment consisted of at least three replicates per test. Differences between groups were assessed using one-way analysis of variance. The statistical significance threshold for all data was p <0.05.

CIH treatment downregulated miR-421 and upregulated TLR4 in rat aortic endothelial cells
The viability of rat aortic endothelial cells was decreased in CIH group ( Figure 1A, p<0.05). Figure 1B showed that CIH group had lower miR-421 levels than control group (p<0.001). However, the mRNA and protein expression of TLR4 were upregulated in CIH group ( Figure 1C and D, p<0.01).

MiR-421 inhibited CIH-induced injury in rat lungs and kidneys
As compared to the sham group, the miR-421 level in the CIH+NC agomiR group was decreased to 51 % (Figure 5 A, p < 0.01), whereas miR-421 level in CIH+miR-421 agomiR group was increased as compared to CIH+NC agomiR group (Figure 5 A, p < 0.001). Rats in the CIH+NC agomiR group had higher mRNA and protein levels of TLR4 than sham group ( Figure 5B and C, p < 0.001 and p < 0.01). However, rats in the CIH+miR-421 agomiR group had lower levels of TLR4 mRNA and protein than CIH+ NC agomiR group (Figures 5 B and C, p <  0.001 and p < 0.05, respectively). HE staining showed that the degree of lung injury and renal injury in the CIH+NC agomiR group was higher than sham group, whereas miR-421 agomiR injection ameliorated CIH-induced lung and renal injury (Figure 5 D). Importantly, miR-421 agomiR attenuated CIH-induced collagen deposition in lung and renal tissues ( Figures 5 D and E).

DISCUSSION
It is well accepted that OSAS is chronic respiratory disease that can induce CIH, which may result in endothelial dysfunction and multiple organ damage [3,4,6]. Many miRNAs, such as miR-664a-3p, miR-630, and miR-30a, play important roles during the development of OSAS [6,13,14]. In this study, CIH-treated endothelial cells had lower viability and decreased miR-421 levels compared to control cells, suggesting that miR-421 may exert protective effects on CIH-treated endothelial cells. To verify it, endothelial cells were transfected with miR-421 mimics and then subjected to CIH. Oxidative stress can be stimulated by CIH to trigger systemic inflammation and endothelial dysfunction [1]. Therefore, cell viability, apoptosis ratio, ROS levels, protein expression levels of apoptosisand inflammation-related genes were assessed. Compared to control group, CIH treatment decreased cell viability, increased cellular ROS levels, promoted apoptosis, caused changes in the protein levels of apoptosis-related genes, and increased inflammatory factor levels. However, miR-421 mimics inhibited these CIH-induced phenomena.
MiRNAs can inhibit transcript expression via complementary base pairing [7]. Previous studies have characterized several direct targets of miR-421, including PDCD4 and caspase-10 in breast cancer [17,22], claudin-11 in gastric cancer [23], and sirtuin 3 in NAFLD [16]. In this study, TargetScan analysis and luciferase reporter analysis identified that TLR4 as a novel miR-421 target. MiR-421mimics suppressed TLR4 mRNA and protein expression in endothelial cells, whereas miR-421 inhibitor promoted TLR4 mRNA and protein expression. This is the first evidence showing that miR-421 targets the TLR4 3′-UTR and regulates the TLR4 expression in endothelial cells.
Consistent with previous reports, TLR4 was upregulated in CIH cells compared to control cells in this study, which suggested that the protective effects of miR-421 on CIH cells may be associated with TLR4 upregulation. To test it, endothelial cells were transfected with miR-421 mimics and TLR4 overexpression plasmid. In vitro experiments showed that co-transfection of TLR4 overexpression plasmid suppressed the protective effects of miR-421on CIH cells. These results suggest that miR-421 protected endothelial cells against CIH-induced injury by targeting TLR4. Conversely, it is well-known that TLR4 may initiate inflammatory responses via at least two major signaling pathways, including myeloid differentiation factor 88 (MyD88)independent and -dependent pathways [24,25]. Further experiments are needed to further determine whether these two TLR4 signaling pathways is involved in the role of miR-421 in CIH.
The rat CIH model was established by imitating the state of OSAS. Consistent with in vitro experiments, miR-421 overexpression inhibited TLR4 mRNA and protein expression in CIH mice compared to control. Moreover, histopathological analysis demonstrated that miR-421 overexpression attenuated CIH-induced damage to the rat lung and kidney.
Thus, miR-421 downregulation and TLR4 upregulation were closely associated with CIHinduced vascular endothelial cells injury. Moreover, miR-421 had protective effects against CIH-induced damage via targeting TLR4. Restoring the function of miR-421 may be a potential strategy for suppressing CIH-induced damage. In addition, these results suggest that TLR4 may be a novel therapeutic target for OSAS.

CONCLUSION
The findings of this study prove that miR-421 downregulation and TLR4 upregulation are closely linked with CIH-induced vascular endothelial cells injury. Moreover, miR-421 protects against CIH-induced vascular endothelial cell injury by targeting TLR4.

DECLARATIONS
analyzed and interpreted the results of the experiments, SQY performed the experiments.

Open Access
This is an Open Access article that uses a funding model which does not charge readers or their institutions for access and distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/ 4.0) and the Budapest Open Access Initiative (http://www.budapestopenaccessinitiative.org/rea d), which permit unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited.