Overexpression of miR-874-3p alleviates LPS-induced apoptosis and inflammation in alveolar epithelial cell by targeting EGR3/NF-κB

1Pediatrics of Integrated Traditional Chinese & Western Medicine, Zhejiang Zhoushan Putuo District Hospital of Traditional Chinese Medicine, Zhoushan, Zhejiang Province, 316100, China; 2Department of Pediatrics, People’s Hospital of Baoshan City, Baoshan, Yunnan Province, 678000, China; 3Department of Pediatrics, People’s Hospital of Baiyun District, Guiyang City, Guizhou Province, 510440, China; 4Department of Infection, Xinhua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, 200092, China


INTRODUCTION
Pneumonia is a common and frequently occurring respiratory disease in pediatrics. It is one of the three most important pediatric diseases and the leading cause of infant mortality worldwide (Alcoba et al., 2017). Pneu-monia is associated with pathogenic factors post infection with bacteria or viruses, and microbial pathogensinduced stimulation of inflammatory response was considered as one of the causes of severe acute pediatrics pneumonia (Qinghe et al., 2019;Zhang et al., 2019). Alveolar epithelial cells are the major targets of inflammatory factors secreted by the infected epithelial cells during the inflammatory response in the lungs (Ito et al., 2015). Damage to alveolar epithelial cells is a key step in the development of pneumonia and ultimately leads to fluid imbalance (Guillot et al., 2013). The state of alveolar epithelial cells contributes to the loss of cellular defense during development of pneumonia (Robb et al., 2016). Therefore, it is important to reduce the inflammation and apoptosis of alveolar epithelial cells during the treatment of pediatric pneumonia.
Dysregulation of microRNAs (miRNAs) has been identified as diagnostic biomarkers for pneumonia (Huang et al., 2017), and miRNAs could mediate expression of target genes involved in immune response during pneumonia (Huang et al., 2018). Therefore, miR-NAs were widely investigated in pediatric pneumonia. Recently, miRNAs have been reported to be implicated in apoptosis and inflammation of alveolar epithelial cells (Li & Liu, 2020). Suppression of apoptosis and inflammation in alveolar epithelial cells by miR-424 could ameliorate acute respiratory distress syndrome (Cheng et al., 2020). MiR-874-3p was reported to increase cell proliferation and suppress apoptosis during cerebral ischemia/reperfusion injury (Jiang et al., 2019), and inhibit inflammatory response during ischemic stroke (Xie et al., 2020). Given that the expression of miR-874-3p was down-regulated in patients with pneumonia in this study, we speculated that miR-874-3p might regulate apoptosis and inflammation of alveolar epithelial cells and play a certain regulatory role in pneumonia development.
EGR3 (early growth response 3) functions as a transcriptional factor to regulate genes involved in inflammation (Xie et al., 2020), cell proliferation (Li et al., 2012) and differentiation (Miao et al., 2017). EGR1 pathway was involved in lung inflammation (Cho et al., 2006), and epithelial barrier disruption (Choi et al., 2012). However, no researches about regulation of EGR in inflammation during pneumonia have not been reported yet. This study was performed to investigate whether miR-874-3p could target EGR3 to regulate apoptosis and inflammation of alveolar epithelial cells.

Serum collection
This study was approved by the Ethics Committee of Zhejiang Zhoushan Putuo District Hospital of Traditional Chinese Medicine. Twenty-seven pediatric patients with pneumonia and nineteen healthy children were recruited at Zhejiang Zhoushan Putuo District Hospital of Traditional Chinese Medicine with written parental informed consents. Patients with anti-inflammatory therapy were excluded from this study, and patients with diagnostic criteria of pneumonia were included in this study. Serum samples were obtained from pneumonia patients and healthy children and stored at -80°C for the functional assays.

Detection of TNF-α and IL-1β
Supernatant was harvested from medium of cell culture through centrifugation at 400×g for 10 minutes. Commercial Elisa kits (Thermo Fisher, Waltham, MA, USA) were used for the detection of TNF-α and IL-1β.

Cell viability and apoptosis assays
HPAEpiC with indicated treatment was seeded for 24 hours followed by LPS incubation. CCK8 solution (Dojindo, Tokyo, Japan) was then added to the wells and incubated at 37°C for 1 hour. Absorbance at 450 nm was measured by Microplate Autoreader (Thermo Fisher). HPAEpiC with indicated treatment and transfection were harvested and resuspended in binding buffer (Invitrogen) with PI (100 µg/mL) and 1 U/ml ribonuclease (Invitrogen) for 30 minutes, and then incubated with fluorescein isothiocyanate-conjugated annexin V (20 µg/ mL) for another 20 minutes. Apoptosis was analyzed by FACS flow cytometer (Attune, Life Technologies, Darmstadt, Germany).

Luciferase reporter assay
3'-UTR of EGR3 or sequence with mutation at the potential binding site between EGR3 and miR-874-3p were subcloned into pGL3 luciferase vector (Promega, Madison, Wisconsin, USA). Co-transfection with EGR3-WT/EGR3-MUT or mimic and inhibitor of miR-874-3p were performed through Lipofectamine 2000. Dual Luciferase Assay Kit (Promega) was used to detect luciferase activities two days later.

RT-qPCR (reverse transcription quantitative real-time PCR)
Total RNAs were isolated from serum samples or HPAEpiC by Trizol (Invitrogen), and reverse-transcribed into cDNAs via PrimeScript RT Master Mix kit (Takara Biotechnology, Dalian, China). TB Green Premix Ex Taq (Takara, Dalian, China) was applied for qRT-PCR analysis. Caenorhabditis elegans miRNA (Cel-miR-39) was used as control to analyze miR-874-3p expression from serum samples. U6 was used as endogenous control to analyze miR-874-3p expression from cells. GAPDH was used as endogenous control to analyze TNF-α, IL-1β and EGR3 expression. Threshold cycle (Ct) value was determined to reflect fluorescent signal of amplification plot, and the relative gene expression was normalized to the controls by the 2 −ΔΔCT method. The primer sequences were shown as below:

Western blot
Total proteins were collected from HPAEpiC by radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology, Beijing, China). Proteins (30 μg) were separated via SDS-PAGE, then transferred anti-β-actin (1:3000; Cell Signaling Technology). After incubation with second antibodies (1:5000; Cell Signaling Technology) for 1 hour, the membranes were conducted with ECL chemiluminescence detection kit (Thermo Fisher), and the blots were visualized and quantified via Quantity One 4.6.2 software (Bio-Rad, Hercules, CA, USA).

Statistical analysis
Data were expressed as mean ± standard deviation. Statistical analyses between different groups were analyzed by GraphPad Prism 5.0 and determined by oneway analysis of variance and Student's t-test. p<0.05 was considered as statistically significant.

Reduced miR-874-3p in serum of pediatric patients with pneumonia
To unravel potential role of miR-874-3p in pediatric pneumonia, serum samples were collected from patients with pneumonia. Data from qRT-PCR demonstrated that miR-874-3p was reduced in serum of pediatric patients with pneumonia compared to the healthy control (Fig. 1A). The relationship between miR-874-3p expression and clinical characteristics of sick children was shown in Table 1. Symptom and signs, including cough, expectoration, dyspnea, and diarrhea, as well as laboratory tests, including white blood cells, neutrophils, and lymphocytes, were significantly associated with miR-874-3p expression (Table 1). HPAEpiC with LPS treatment was used to establish in vitro cell model of pneumonia. A dose-dependent reduction of miR-874-3p was identified in LPS-treated HPAEpiC (Fig.  1B), suggesting a potential role of miR-874-3p in pediatric pneumonia.

DISCUSSION
Alveolar epithelial cells are the prime sites of bacterial or viral infection, and the infection could result in apoptosis and cytotoxicity of the epithelial cells (Herold et al., 2008). Prevention of apoptosis and cytotoxicity of alveolar epithelial cells could help ameliorate pneumonia (Kosmider et al., 2012). MiRNAs, with regulatory ability in cellular senescence, have been shown to be implicated in apoptosis of alveolar epithelial cells (Adameova et al., 2018). Since miR-874 has been reported to suppress cell proliferation and promote non-small cell lung cancer cell apoptosis (Wang et al., 2020), the potential regulatory roles of miR-874-3p in cytotoxicity of alveolar epithelial cells and development of pneumonia were evaluated in this study. MiR-874-3p was down-regulated in serum of patients with pneumonia and LPS-treated HPAEpiC. LPS, as an endotoxin, could stimulate apoptosis and cytotoxicity. Therefore, LPS was widely used as a stimulatory model of acute pneumonia (Zhang et al., 2020). Our results indicated that LPS treatment reduced viability of HPAEpiC and enhanced apoptosis. Moreover, LPS could stimulate inflammatory response and promote pathogenesis of pneumonia (Meng et al., 2012). Proinflammatory factors TNF-α and IL-1β were found to be up-regulated in HPAEpiC post LPS treatment during this study. Alleviation of LPS-induced cytotoxicity and inflammation could help improve acute pneumonia (Zhang et al., 2019). We therefore validated the role of miR-874-3p in LPS-induced inflammation and apoptosis of HPAEpiC.
In line with previous study that miR-874 suppressed cell proliferation and promoted non-small cell lung cancer cell apoptosis (Wang et al., 2020), knockdown of miR-874-3p aggravated LPS-induced decrease of cell viability and increase of apoptosis in HPAEpiC. Moreover, LPS-induced secretion of TNF-α and IL-1β was also repressed by miR-874-3p over-expression, consistent with previous study that miR-874 could attenuate inflammatory response with decreased IL-1β and TNF-α in diabetic nephropathy (Yao et al., 2019). These results suggested that miR-874-3p could alleviate LPS-induced inflammation and apoptosis in alveolar epithelial cells, thus ameliorating development of pediatric pneumonia. Animal model of pneumonia should be established to further investigate the role of miR-874-3p in pediatric pneumonia.

CONCLUSION
MiR-874-3p was reduced in serum of pediatric patients with pneumonia. We found that miR-874-3p could promote viability of LPS-treated alveolar epithelial cells, while suppressing apoptosis and inflammation through targeting the EGR3/NF-κB pathway. This study provided a potential therapeutic target for pediatric pneumonia.