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Kcnq1ot1/miR-381-3p/ETS2 Axis Regulates Inflammation in Mouse Models of Acute Respiratory Distress Syndrome

Inflammatory mediators play a key role in the pathogenesis of acute respiratory distress syndrome (ARDS). In this study, we aimed to explore the involvement of the Kcnq1 opposite strand/antisense transcript 1 (Kcnq1ot1)/miR-381-3p/E26 transformation-specific proto-oncogene 2 (ETS2) axis in inflammation of lipopolysaccharide (LPS)-induced ARDS. Microarray analysis revealed ETS2 as an upregulated gene in ARDS. Then, a LPS-induced ARDS mouse model was constructed, with a series of gain- or loss-of-function experiments conducted to evaluate the lung function and neutrophil extracellular trap (NET) formation in lung tissue and determine the neutrophil number, myeloperoxidase (MPO) activity, and inflammatory factor levels in bronchoalveolar lavage fluid (BALF). As the results revealed, downregulated expression of ETS2 resulted in improved lung function, decreased NETs, MPO activity, and levels of interleukin (IL)-6 and tumor necrosis factor alpha (TNF-α), as well as increased IL-10 level. Then, the assays of dual-luciferase reporter, RNA-binding protein immunoprecipitation (RIP), and RNA pull-down were performed to validate that Kcnq1ot1 promoted ETS2 expression by competitively binding to miR-381-3p. Meanwhile, it was also found that Kcnq1ot1 silencing reversed the promotive effect of EST2 on ARDS. Our results provide evidence that Kcnq1ot1 silencing may reduce the inflammatory response in LPS-induced ARDS via inhibition of miR-381-30-dependent ETS2, thereby presenting new molecular understanding for the development of ARDS.


INTRODUCTION
Acute respiratory distress syndrome (ARDS) is a major clinical presentation of acute lung injury, characterized by decreased lung compliance, noncardiogenic pulmonary edema, and refractory hypoxia. 1 Severe ARDS remains deadly, with a mortality rate of approximately 40%, despite advancements in supportive care. 2 ARDS develops as an inflammatory process in response to extrapulmonary and pulmonary stimuli to the alveolar-capillary membrane, leading to increased vascular permeability and subsequently pulmonary edema. 3 Pulmonary neutrophilic inflammation is a principal process triggering ARDS during microbial infection in the alveolar microenvironment. 4,5 Furthermore, elevation of neutrophil recruitment, myeloperoxidase (MPO) activity, and reactive oxygen species (ROS) found in lungs from ARDS mice favors the formation of neutrophil extracellular traps (NETs). 6 NETs, representing a danger-associated molecular pattern, associate with inflammatory reactions and tissue injury in the lung. 7 Therefore, targeting NETs or a danger-associated molecular pattern may be a novel therapeutic direction for the treatment of ARDS. 7 E26 transformation-specific proto-oncogene 2 (ETS2), a transcription factor in the ETS family, is a direct effector in the regulation of macrophage functions during inflammatory responses. 8 As a critical substrate of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway, ETS2 modulates multiple genes induced by MAPK activation during tumor development. 9,10 ETS2 is a target of miR-381-3p, and the MAPK/miR-381 axis is involved in epithelial-mesenchymal transition in lung cancer. 11 More importantly, miR-381 is involved in the treatment of lipopolysaccharide (LPS)-induced acute lung injury by dexmedetomidine. 12 The same study also predicted that miR-381-3p was regulated by long non-coding RNA (lncRNA) Kcnq1 opposite strand/antisense transcript 1 (Kcnq1ot1) through competitive binding. Kcnq1ot1, located at human chromosome 11p15.5, 13 is a 91-kb-long RNA polymerase II-encoded nuclear transcript. 14 The stability of Kcnq1ot1 is critical for bidirectional gene silencing. 14 lncRNA Kcnq1ot1 is expressed at a high level in lung adenocarcinoma and acts as an oncogene to promote malignancy and chemoresistance in cancer cells. 15 Despite previous studies showing the involvement of miR-381-3p and Kcnq1ot1 in various lung diseases, their impact in ARDS is currently unknown. Therefore, we aimed to investigate the role of the Kcnq1ot1/miR-381-3p/ETS2 axis in neutrophilic inflammation during ARDS.

ETS2 Is Involved in LPS-Induced ARDS
We first performed a differential gene expression analysis on an ARDS-related expression dataset, GSE76293. A heatmap illustrating the most differentially expressed genes is shown in Figure 1A and Table S1. Among all of the obtained differentially expressed genes, we noticed that the p value of the ETS2 gene was the smallest, and it was significantly highly expressed in ARDS. Therefore, the significantly upregulated ETS2 gene was identified as the target gene following differential analysis of the microarray. To explore the role of ETS2 in ARDS, we developed a mouse model. The ARDS mouse models induced by LPS for 48 h had a decreased ratio of the partial pressure of arterial oxygen (PaO 2 )/percentage of inspired oxygen (FiO 2 ) ( Figure 1B), increased lung wet weight to dry weight ratio (Figure 1C), reduced number of alveolar sacs ( Figures 1D and 1E), and increased crowded score ( Figure 1F) when compared to saline-treated controls. These results demonstrated the successful establishment of the ARDS mouse model.
The expression of ETS2 in peripheral blood was then detected by qRT-PCR and western blot analysis. Expression of ETS2 mRNA and protein was significantly increased in ARDS mice versus control  The measurement data are presented as mean ± SD. Comparison between two groups was analyzed by an unpaired t test, Welch's correction t test, and a Mann-Whitney test. n = 8.
mice ( Figures 1G and 1H, p < 0.05), thereby validating our bioinformatic findings. To study the effects of genetic manipulation of ETS2, we then silenced ETS2 in ARDS mice by treatment with short hairpin RNA (shRNA), which predictably resulted in a reduced expression of ETS2 in peripheral blood when compared with those treated with sh-negative control (NC) ( Figures 1G and 1H, p < 0.05). Encouragingly, however, sh-ETS2 treatment also increased the ratio of PaO 2 /FiO 2 ( Figure 1B, p < 0.05) and decreased the lung wet weight to dry weight ratio when compared to sh-NC ( Figure 1C, p < 0.05). A significantly increased number of alveolar sacs, as well as a significantly decreased crowded score, was observed in ARDS mice treated with sh-ETS2 versus sh-NC treatment ( Figures 1D-1F, p < 0.05). Taken together, these results indicate that ETS2 is involved in the pathogenesis of LPS-induced ARDS.

Downregulated ETS2 Reduces Neutrophilic Inflammation in Lungs of ARDS Mouse Models
Having identified an involvement of ETS2 in ARDS mouse models, we continued to explore its effects on neutrophilic inflammation in the lung. The total cells and neutrophils were significantly recruited in the bronchoalveolar lavage fluid (BALF) of the ARDS mouse models, which were reduced in response to the sh-ETS2 treatment ( Figure 2A, p < 0.05). At the same time, we found that the MPO activity and the levels of tumor necrosis factor alpha (TNF-a) and interleukin (IL)-6 in the BALF of the ARDS mouse models were significantly increased, accompanied by decreased IL-10, and these changes were reversed in the presence of sh-ETS2 treatment ( Figures  2B and 2C, p < 0.05). MPO and histone are known biomarkers of NETs to identify neutrophilic inflammation. Indeed, when we performed an immunofluorescence assay, we found significantly increased NETs in the lung tissue of ARDS mouse models, and we showed that they could be diminished by treatment with sh-ETS2 ( Figure 2D).
Kcnq1ot1 Competitively Binds to miR-381-3p to Promote ETS2 Expression Through the DIANA Tools dataset, we further found that miR-381-3p had binding sites for Kcnq1ot1 ( Figure 4A). Luciferase activity was significantly reduced when miR-381-3p was co-transfected with Kcnq1ot1-WT versus agomir-NC ( Figure 4B, p < 0.05). We also observed that mRNA expression of Kcnq1ot1 in BALF neutrophils from ARDS mice was significantly increased when compared to control mice ( Figure 4C, p < 0.05). RNA-binding protein immunoprecipitation (RIP) ( Figure 4D) and RNA pull-down ( Figure 4E) assays confirmed that Kcnq1ot1 indeed bound to miR-381-3p. We then designed two Kcnq1ot1 shRNAs to knock down Kcnq1ot1 in BALF neutrophils from ARDS mice. Silencing of Kcnq1ot1 in neutrophils resulted in a significant increase in mRNA expression of miR-381-3p, and a significant decrease in the expression of Kcnq1ot1 and ETS2, when compared to sh-NC ( Figure 4F, p < 0.05). Taken together, these results confirm that Kcnq1ot1 competitively binds to miR-381-3p to promote ETS2 expression.

Kcnq1ot1/miR-381-3p/ETS2 Axis Regulates Inflammatory Response in ARDS Mouse Models
Having identified that Kcnq1ot1 regulates ETS2 expression through miR-381-3p, we focused our attention on further analyzing the role of the Kcnq1ot1/miR-381-3p/ETS2 axis in the inflammatory response in ARDS. Lung function tests revealed that the ratio of PaO 2 /FiO 2 increased while the lung wet weight to dry weight ratio decreased in response to treatment of sh-Kcnq1ot1 + antagomir-NC, versus treatment with sh-NC + antagomir-NC. However, the ratio of PaO 2 /FiO 2 and the lung wet weight to dry weight ratio induced by sh-Kcnq1ot1 + antagomir-NC were significantly decreased and increased, respectively, by treatment with the sh-Kcnq1ot1 + miR-381-3p antagomir. The ratio of PaO 2 /FiO 2 was decreased while the lung wet weight to dry weight ratio was increased in response to sh-Kcnq1ot1 + ETS2, in comparison to treatment with sh-Kcnq1ot1 + vector-NC (Figures 5A and 5B, p < 0.05).
H&E staining of lung sections showed that the number of alveolar sacs was significantly increased, accompanied by lower lung parenchyma score in the presence of sh-Kcnq1ot1 + antagomir-NC, when compared to sh-NC + antagomir-NC. In addition, relative to sh-Kcnq1ot1 + antagomir-NC, treatment with sh-Kcnq1ot1 + miR-381-3p antagomir resulted in a decreased number of alveolar sacs and an increased lung parenchyma score. We also observed a reduced number of alveolar sacs and elevated lung parenchymal score in response to sh-Kcnq1ot1 + ETS2, versus sh-Kcnq1ot1 + vector-NC (Figures 5C-5E, p < 0.05).
Next, we measured the number of neutrophils and found that total cells and neutrophils in the BALF treated with sh-Kcnq1ot1 + antagomir-NC www.moleculartherapy.org were decreased compared to those treated with sh-NC + antagomir-NC. However, in contrast to treatment with sh-Kcnq1ot1 + antagomir-NC, the total cells and neutrophils in BALF with sh-Kcnq1ot1 + miR-381-3p antagomir treatment were increased. When compared with sh-Kcnq1ot1 + vector-NC treatment, both the total cells and neutrophils in BALF with sh-Kcnq1ot1 + ETS2 treatment were significantly  enriched ( Figure 5F, p < 0.05). At the same time, we found that compared with sh-NC + antagomir-NC treatment, the MPO activity in BALF treated by sh-Kcnq1ot1 + antagomir-NC was decreased, and the levels of TNF-a and IL-6 were decreased, accompanied by increased IL-10. Similarly, relative to the sh-Kcnq1ot1 + antagomir-NC treatment group, the BALF treated with sh-Kcnq1ot1 + miR-381-3p antagomir showed increased MPO activity and levels of TNF-a and IL-6, and a decreased level of IL-10. Additionally, in contrast to sh-Kcnq1ot1 + vector-NC treatment, the MPO activity and levels of TNF-a and IL-6 were increased, accompanied by decreased IL-10, in the BALF treated with sh-Kcnq1ot1 + ETS2 ( Figures 5G and 5H, p < 0.05). Furthermore, as the immunofluorescence assay showed, compared to treatment with sh-NC + antagomir-NC, the presence of sh-Kcnq1ot1 + antagomir-NC resulted in reduced NETs in lung tissue of mice, and treatment with sh-Kcnq1ot1 + miR-381-3p antagomir displayed more NETs in lung tissue than did treatment with sh-Kcnq1ot1 + antagomir-NC. The sh-Kcnq1ot1 + ETS2 treatment group showed more NETs in the lung tissue than did the group treated with sh-Kcnq1ot1 + vector-NC ( Figure 5I). Taken together, the aforementioned results suggest that the Kcnq1ot1/miR-381-3p/ETS2 axis could regulate the inflammatory response in ARDS mouse models.

DISCUSSION
ARDS is a high-incidence and sometimes lifethreatening syndrome, and early diagnosis and treatment are therefore important for better outcomes. 16 Excessive neutrophilic inflammation has been shown to have a critical role in various lung conditions, 17,18 but its mechanism is largely unknown in the context of ARDS. lncRNA Kcnq1ot1 is involved in modulating imprinted genes in the Kcnq1 domain and facilitates the progression of non-small cell lung cancer through the miR-27b-3p/HSP90AA1 axis as an oncogene. 19 In this study, we uncover an important novel finding that the Kcnq1ot1/miR-381-3p/ETS2 axis regulates the inflammatory response in LPS-induced ARDS mice (a schematic diagram is shown in Figure 6).
We first identified ETS2 as the most significantly upregulated gene in ARDS according to microarray analysis of the ARDS-related GSE76293 expression dataset, and we validated that ETS2 indeed participated in the progression of LPS-induced ARDS. This result agrees with previous studies that ETS2 is involved in the inflammatory response in lung diseases. For example, ETS2 has been implicated in pulmonary fibrosis 20 and shown to elevate the expression of granulocyte-macrophage colony-stimulating factor in non-small lung carcinoma cells. 21 Neutrophil recruitment is a critical process in response to bacterial infections in the early stage, but excessive and inappropriate recruitment results in tissue damage. 4 In the current study, we found that downregulation of ETS2 reduced neutrophil accumulation, MPO activity, NET formation, TNF-a, and IL-6 levels. ETS2 downregulation may enhance neutrophil recruitment in ARDS www.moleculartherapy.org mice on the grounds that a previous study showed that alleviation of lung injury was commonly associated with increased NET formation induced by pulmonary neutrophil recruitment. 7 Moreover, ETS2 downregulation may reduce inflammation because activated neutrophils produce proinflammatory cytokines such as TNF-a and ILs. 1 Our result also agrees with a previous study that ETS2 is involved in the modulation of inflammatory cytokines including ILs and TNF-a. 22 Collectively, our results strongly suggest that ETS2 is involved in acute neutrophil inflammation, which is one of the hallmarks of ARDS. 3 Target genes of miR-381-3p are known to be involved in cellular processes and cellular macromolecule metabolism. 23 Notably, miR-381-3p mediates antigen-presenting ability in dendritic cells and anti-tuberculosis cellular immune responses by targeting CD1c. 24 In this study, we revealed that miR-381-3p directly targeted 3 0 UTR of ETS2 and reduced the expression of ETS2. Therefore, miR-381-3p may inhibit inflammation in LPS-induced ARDS mice by targeting ETS2.
Furthermore, we found that Kcnq1ot1 bound to miR-381-3p to promote ETS2 expression. Kcnq1ot1 was demonstrated to serve as a competing endogenous (ceRNA) for miRNAs, such as sponging miR-214-3p to regulate caspase-1. 25 Inhibition of Kcnq1ot1 reduces inflammation in myocardial ischemia/reperfusion injury by targeting AdipoR1 via the p38 MAPK/nuclear factor kB (NF-kB) signaling pathway. 26,27 These results from previous studies agree with the idea that lncRNA Kcnq1ot1 promotes inflammatory response in ARDS mice. Our results further suggested that the effect of Kcnq1ot1 may work through sponging miR-381-3p and enhancing ETS2 expression. Conversely, Kcnq1ot1 silencing may attenuate the inflammatory response in ARDS.
In conclusion, we provide evidence that the Kcnq1ot1/miR-381-3p/ ETS2 axis regulates neutrophil inflammatory response in LPSinduced ARDS mice. This study identifies the Kcnq1ot1/miR-381-3p/ETS2 axis as a novel biomarker for ARDS, which will ultimately provide new insights for prediction, prognosis, and treatment for patients with ARDS.

Ethics Statement
All animal procedures were conducted in accordance with the Guide for the Care and Use of Laboratory Animals, published by the NIH (publication no. 85-23, revised 1996). 28 All animal experiments were approved by the Animal Care Committee at Zhejiang Provincial People's Hospital, People's Hospital of Hangzhou Medical College.
In Silico Analysis ARDS-related expression dataset GSE76293 and its annotation probe were obtained through the GEO database (GEO: GSE76293). Differential gene expression analysis was performed using the R software. Differentially expressed genes were defined as |log 2 fold change (FC)| > 1 and p < 0.05.  The measurement data are presented as mean ± SD. Comparison between two groups was analyzed by an unpaired t test. Comparisons among multiple groups were performed with one-way ANOVA, followed by Tukey's post hoc test. n = 8. The cell experiments were repeated three times.  www.moleculartherapy.org

Mouse Models Simulating ARDS
In total, 74 male specific pathogen-free (SPF) C57BL/6 mice (12 weeks old, weighing 18-23 g) were purchased from the Animal Experimental Research Center, Zhejiang Chinese Medical University (Hangzhou, Zhejiang, China). The mice were acclimatized for 1 week before they were weighed. 66 mice were randomly selected to establish models of ARDS. The mice were anesthetized by intraperitoneal injection of sodium pentobarbital (50 mg/kg, WS20060401, Sinopharm Chemical, China). Then, mice were fixed in the supine position to expose the skin of the neck, which was disinfected with 75% ethanol. The skin was cut open to separate the subcutaneous tissue layer by layer and expose the trachea. A micro-syringe was inserted into the trachea through the tracheal cartilage ring gap toward the heart end. If there was no resistance to pull back, 5 mg/kg LPS (Escherichia coli O111:B4 dissolved in saline, Sigma, St. Louis, MO, USA) was injected. Immediately after the operation, the mice were set upright and rotated, so that the LPS was evenly distributed in the lungs. The mice were supplied with oxygen, and the oxygen concentration was maintained at 2 L/min until they were awake. Then they were returned to the cage for routine feeding. There were 56 successfully established mouse models, with a success rate of 84.85%. 29,30 Animal Grouping Mice were randomly assigned into eight groups (n = 8 per group): control group (intratracheal instillation of 0.9% normal saline); ARDS group (intratracheal instillation of 5 mg/kg LPS); sh-NC group (intratracheal instillation of 5 mg/kg LPS 1 h before intravenous injection of sh-NC through caudal veins); sh-ETS2 group (5 0 -AGAAAU GGAAUCCAAGCCUGUUGGC-3 0 ; 5 0 -GCCAACAGGCUUGGAU UCC AUUUCU-3 0 ) (intratracheal instillation of 5 mg/kg LPS 1 h before intravenous injection of sh-ETS2 through caudal veins); sh-Kcnq1ot1 (5 0 -ACCCAACAACTCTGGAACATA-3 0 ; 5 0 -TATGTT CCAGAGTTGTTGGG-3 0 ) + antagomir-NC group (intratracheal instillation of 5 mg/kg LPS 1 h before intravenous injection of sh-Kcnq1ot1 + antagomir-NC through caudal veins); sh-Kcnq1ot1 + miR-381-3p antagomir group (intratracheal instillation of 5 mg/kg LPS 1 h before intravenous injection of sh-Kcnq1ot1 + miR-381-3p antagomir through caudal veins); sh-Kcnq1ot1 + vector-NC group (intratracheal instillation of 5 mg/kg LPS 1 h before intravenous injection of sh-Kcnq1ot1 + vector-NC through caudal veins); and sh-Kcnq1ot1 + ETS2 group (intratracheal instillation of 5 mg/kg LPS 1 h before intravenous injection of sh-Kcnq1ot1 + ETS2 through caudal veins). Lentiviruses of sh-Kcnq1ot1, sh-ETS2, and overexpressed ETS2 were purchased from Shanghai GenePharma (Shanghai, China). Lentivirus (1 Â 10 9 transducing units [TU]) was dissolved in 50 mL of normal saline and then injected through the caudal vein. 31 miR-381-3p antagomir (miR30017081-4-5) and antagomir-NC (miR3N0000002-4-5) (100 nmol/L) were injected through the caudal vein (Guangzhou RiboBio, Guangzhou, Guangdong, China). Blood samples were collected from abdominal aorta 48 h after LPS injection. In addition, BALF was collected by bronchoalveolar lavage (BAL) in all groups. 29,30 Total and differential white blood cell counts were determined in BALF by an automated hematology analyzer (Sysmex, Kobe, Japan). 32 Arterial Blood Gas Analysis and Lung Wet Weight to Dry Weight Ratio PaO 2 and PaCO 2 were measured using an automatic blood gas analyzer (ABL800 FLEX, Radiometer, Bronshoj, Denmark). The oxygenation index was calculated by the ratio of PaO 2 to FiO 2 . Lungs were excised and weighed for wet weight immediately after the mice were sacrificed. Lungs were then dehydrated in an oven at 60 C, and the dry weight was measured 72 h later. The ratio of dry weight to wet weight of the lung was then calculated. 32

Histological Evaluation of Lung Injury
Lower right lung lobe was cut into thin sections (5 mm). The number of alveolar sacs was determined after H&E staining by investigators blinded to experimental groups. Three lung sections of each mouse were evaluated. Three high-power fields (Â100) were randomly selected in each section for observation. The average number of alveolar sacs per mouse was determined by the sum from all fields divided by 9. The crowded area was defined as thickened septa in lung parenchyma where alveolar sacs were partly or completely collapsed under H&E-stained sections. A scoring system was used to quantify the crowded area by investigators blinded to experimental groups, defined as follows: 0, no crowded area; 1, 15% of the total high-power field was a crowded area; 2, 15%-25% crowded area; 3, 25%-50% crowded area; 4, 50%-75% crowded area; 5, R75%-100% of the area was crowded. 28 where DDCt = DCt experimental group À DCt control group and DCt = Ct target gene À Ct internal reference gene . The Ct value refers to the fractional cycle at which the fluorescence intensity equals the threshold fluorescence, at which point the amplification is in logarithmic growth. The experiment was repeated three times. 25

Western Blot Analysis
An equal amount (10-30 mg) of protein extract (from peripheral blood or neutrophils) was separated by SDS-PAGE with gradient acrylamide (8%-10%). After electrophoresis, separated proteins were electrophoretically transferred to a polyvinylidene fluoride (PVDF) membrane (Amersham Biosciences, Amersham, UK). Nonspecific binding sites in the PVDF membrane were blocked. Then, membranes were incubated with rabbit anti-EST2 (1 mg/mL, ab153652) overnight at 4 C, followed by incubation with HRP-labeled goat anti-rabbit secondary antibody (1:5,000, ab6721) for 1 h. An enhanced chemiluminescence detection kit (BB-3501, Amerham) was used to visualize images, which were obtained with a Bio-Rad image analysis system (Bio-Rad, Hercules, CA, USA) and analyzed by Quantity One v4.6.2 software. Relative protein level was determined as the gray value of the corresponding protein band divided by the gray value of the b-actin protein band. The experiment was repeated three times.

RIP Assay
Neutrophils were collected after trypsinization. Lysis buffer containing RNase (Life Technologies, Gaithersburg, MD, USA) and protease inhibitor cocktail (Roche) was added to lyse cells. The lysate was centrifuged at 12,000 Â g for 30 min and supernatant was collected. Protein G-agarose beads were incubated with Ago2 antibody (P10502500, Otwo Biotech, Shenzhen, Guangdong, China) and IgG (Sigma, St. Louis, MO, USA) for 2 h at 4 C. Then, cell lysate supernatant was added and incubated overnight at 4 C. RNA was extracted from magnetic beads using TRIzol reagent (Invitrogen). mRNA expressions of Kcnq1ot1 and miR-381-3p were detected by qRT-PCR.

RNA Pull-Down Assay
Neutrophils were transfected with 50 nM biotinylated bio-miR-381-3p-WT, bio-miR-381-3p-bio-mut, or the corresponding NC-bio. After 48 h, harvested cells were incubated in a specific lysis buffer (Ambion) for 10 min and centrifuged at 14,000 Â g. Supernatant was collected and incubated with M-280 streptavidin magnetic beads (S3762, Sigma) pre-coated with RNase-free BSA and yeast tRNA (TRNABAK-RO, Sigma, St. Louis, MO, USA) for 3 h at 4 C. Beads were then washed twice with pre-chilled lysis buffer, three times with low-salt buffer, and once with high-salt buffer. Bound RNA was purified by TRIzol. mRNA expression of Kcnq1ot1 was detected by qRT-PCR.

Statistical Analysis
Statistical analysis was performed using SPSS version 21.0 (IBM Software, New York, NY, USA). The measurement data were presented as mean ± SD. The normal distribution was checked by a Kolmogorov-Smirnov test, and the homogeneity of variance was checked by Levene's test. For comparisons between two groups, an unpaired t test was performed when data were in line with normal distribution and homogeneity of variance, while a Welch's correction t test was conducted when data were consistent with normal distribution yet inconsistent with homogeneity of variance, and a Mann-Whitney test was carried out for data with skewed distribution. For compari-sons among multiple groups, one-way ANOVA with a Tukey's post hoc test was performed when data conformed to normal distribution and homogeneity of variance, while Brown-Forsythe's and Welch's ANOVA tests were conducted when data were in accordance with normal distribution yet disobeyed from homogeneity of variance (Tamhane T2 for pairwise comparisons), and a Kruskal-Wallis test was used for data with skewed distribution (Dunn's multiple comparisons test for pairwise comparisons). Statistical significance was defined as p < 0.05.

AUTHOR CONTRIBUTIONS
X.J., T.Z., L.L., and X.C. and designed the study. M.Y., Q.L., D.W., and R.S. collated the data, carried out data analyses, and produced the initial draft of the manuscript. X.J. and M.Y. contributed to drafting the manuscript. All authors have read and approved the final submitted manuscript.

CONFLICTS OF INTEREST
The authors declare no competing interests.