The lncRNA HOTAIR regulates autophagy and affects lipopolysaccharide‐induced acute lung injury through the miR‐17‐5p/ATG2/ATG7/ATG16 axis

Abstract Long non‐coding ribonucleic acids (lncRNAs) play critical roles in acute lung injury (ALI). We aimed to explore the involvement of lncRNA HOX transcript antisense intergenic ribonucleic acid (HOTAIR) in regulating autophagy in lipopolysaccharide (LPS)‐induced ALI. We obtained 1289 differentially expressed lncRNAs or messenger RNAs (mRNAs) via microarray analysis. HOTAIR was significantly upregulated in the LPS stimulation experimental group. HOTAIR knockdown (si‐HOTAIR) promoted cell proliferation in LPS‐stimulated A549 and BEAS‐2B cells, suppressing the protein expression of autophagy marker light chain 3B and Beclin‐1. Inhibition of HOTAIR suppressed LPS‐induced cell autophagy, apoptosis and arrested cells in the G0/G1 phase prior to S phase entry. Further, si‐HOTAIR alleviated LPS‐induced lung injury in vivo. We predicted the micro‐ribonucleic acid miR‐17‐5p to target HOTAIR and confirmed this via RNA pull‐down and dual luciferase reporter assays. miR‐17‐5p inhibitor treatment reversed the HOTAIR‐mediated effects on autophagy, apoptosis, cell proliferation and cell cycle. Finally, we predicted autophagy‐related genes (ATGs) ATG2, ATG7 and ATG16 as targets of miR‐17‐5p, which reversed their HOTAIR‐mediated protein upregulation in LPS‐stimulated A549 and BEAS‐2B cells. Taken together, our results indicate that HOTAIR regulated apoptosis, the cell cycle, proliferation and autophagy through the miR‐17‐5p/ATG2/ATG7/ATG16 axis, thus driving LPS‐induced ALI.

Ad12-SV40 2B (BEAS-2B) cells. Therefore, LPS-stimulated A549 and BEAS-2B cells have emerged as clinically relevant models of ALI. 3,4 Autophagy is an evolutionarily conserved degradation pathway responsible for delivering cytoplasmic components to the lysosome in vesicles called autophagosomes. 5 Autophagosome formation depends on several autophagy-related genes (ATGs), including light chain 3B (LC3B) and Beclin-1. 6 Autophagy inhibition is known to ameliorate LPSinduced ALI. For instance, Fu et al 7 found that hydrogen-rich saline inhibited both LPS-induced ALI and endothelial dysfunction by regulating autophagy. Likewise, Chen et al 8 reported that miR-100 from microvesicles enhanced autophagy and ameliorated ALI. These studies indicate that autophagy is a potential therapeutic target in ALI, warranting further investigation.
Micro-ribonucleic acids (miRNAs) are a class of small non-coding RNAs (ncRNAs) that are ~22 nucleotides (nt) long and regulate messenger (mRNA) as well as long non-coding RNA (lncRNA) expression at the post-transcriptional level via miRNA binding sites. 9,10 MiRNAs, the study of which has become a research hotspot within molecular biology, are reported to play important regulatory roles in ALI pathogenesis, progression and treatment. 11,12 For instance, Neudecker et al 13 found that the transfer of miR-223 from neutrophils to lung ECs dampens ALI in mice. Jansing et al 12 reported that miR-21-KO alleviates alveolar structure remodelling and inflammatory signalling in ALI. These studies suggest that miRNAs may serve as promising targets for the prevention and treatment of ALI. Even though miR-NAs have been studied for decades, those involved in autophagy regulation have only recently received attention. Zhou et al 14 observed that mesenchymal stem cells could alleviate LPS-induced ALI in mice via miR-142a-5p-regulated pulmonary EC autophagy. Therefore, our current study aimed to explore novel miRNAs that induce autophagy in ALI.
LncRNAs are defined as transcripts of >200 nt in length without protein-coding potential. Further, these can alter miRNA expression by acting as competing endogenous RNAs and can interact with translation machinery by targeting mRNA. 15,16 Different researchers have reported lncRNAs to have a variable influence on ALI over the past few years. Wang et al 17 found that lncRNAs were significantly altered in LPS-induced ALI and that targeting lncRNA could suppress the LPS-induced inflammatory response. Liao et al 18 found that lncRNA maternally expressed gene 3 (MEG3) could adsorb miRNA-7B to regulate nucleotide-binding oligomerization domain as well as leucine rich repeat and pyrin domain-containing 3, thus suppressing LPS-induced ALI. Studies have shown that lncRNAs can inhibit downstream-related signal transduction through the miRNA/mRNA axis, reduce cell autophagy and alleviate ALI. 19,20 However, the exact mechanism through which lncRNAs regulate autophagy to induce ALI via the adsorption of miRNAs remains unknown.
In this study, we assessed the biological function of lncRNA HOTAIR and miR-17-5p as well as their effects on cell proliferation and apoptosis. Furthermore, we explored the regulatory network involving HOTAIR, miR-17-5p and autophagy to open new avenues for the treatment and diagnosis of ALI.

| Microarray analysis of differentially expressed miRNAs
We downloaded raw gene expression data from the US National Center

| Pathway enrichment analysis
We performed pathway enrichment analysis on the differentially expressed mRNAs using the Kyoto Encyclopedia of Genes and Genomes (KEGG) and the R software package clusterProfiler version 3.10.1 (https://guang chuan gyu.github.io/softw are/clust erPro filer/).

| Animals
All animal experiments were approved by the Animal Care and Use

| ALI mouse model
We randomly divided 24 C57BL/6 mice into four groups: a sham operation group, a model group, a lentivirus (LV)-control group and a LV-si-HOTAIR group (n = 6 per group). To establish the ALI model, we anaesthetized mice with intraperitoneal injections of 1% pentobarbital sodium (50 mg/kg). Mice were endotracheally intubated with an indwelling needle. Using a 1-mL syringe, we pushed 10 μg LPS in 50 μL phosphate-buffered saline (PBS) into the tube. The sham group received an equal volume of PBS. 4 We injected control and si-HOTAIR lentiviruses (2 × 10 8 TU/mL; Hanbio) through the tail vein 30 minutes before LPS stimulation. After 6 hours of stimulation, mice were killed, and lung tissues were removed and stored at −80°C.

| Haematoxylin and eosin (H&E) staining
We placed lung tissue in 10% formalin overnight, dehydrated it and embedded it in paraffin. The tissue was sliced into 5-mm thick sections, fixed on a glass slide, dried and then dyed using HE staining solution (Solarbio) as per the manufacturer's instructions. We soaked the slices in xylene, in gradient-concentration ethanol and then in haematoxylin before sealing them with resin. After drying, we observed changes to the alveolae and alveolar interstitial structure in lung tissue sections, photographing them under a light microscope. bound RNAs were then subjected to RT-qPCR for quantification and analysis as described above.

| Transmission electron microscopy
We observed autophagy in A549 and BEAS-2B cells under a transmission electron microscope. Cells from each experimental group were collected, digested with 2.5 g/L trypsin, centrifuged at 1000 g, washed with PBS, and collected in microcentrifuge tubes. We then fixed the cells with 25 g/L glutaraldehyde plus 10 g/L citric acid. After dehydration with graded ethanol and infiltration, cells were embedded in epoxy resin. We sliced the resin using an ultramicrotome, stained the cells with uranyl acetate as well as lead citrate and observed them via transmission electron microscopy (TEM).

| Real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR)
We extracted total RNA using TRIzol reagent (Invitrogen). The extracted RNA was reverse-transcribed into complementary deoxyribonucleic acid (cDNA) using a PrimeScript RT Reagent Kit (TaKaRa) as per the manufacturer's instructions. We performed RT-qPCR using an ABI 7500 system (Applied Biosystems) and a SYBR Premix ExTaq II kit (TaKaRa). The primers for HOTAIR, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), miR-17-5p, and U6 were as follows:

| Transient transfection and dual luciferase reporter assay
Cells were seeded into 96-well plates 1 day before transfection. We Reporter Assay System (Promega) as per the manufacturer's instructions. All assays were independently performed in triplicate.

| Western blot
Cells were harvested and lysed using ice-cold lysis buffer (Beyotime Institute of Biotechnology), and protein concentration was determined using a bicinchoninic acid protein assay kit (Keygentec). We separated denatured proteins (20 μg) by sodium dodecyl sulphate polyacrylamide gel electrophoresis and transferred them onto poly-

| Cell proliferation assay
We prepared single-cell suspensions via trypsinization and seeded the indicated cell lines into six-well plates at a density of 500 cells/ well. After 2 weeks of culture, the cells were digested with trypsin.
The 10-μL cell suspension was mixed well with 10 μL Phenol Blue and added to the counting plate. After leaving the mixture at room temperature for 3 minutes, we observed and counted cells under an inverted microscope. The remaining cells were then inoculated in 96-well plates at a density of 3 × 10 3 and cultured for 24-72 hours.
We detected the optical density at 450 nm every 24 hours using a CCK-8 kit as per the manufacturer's instructions. Each experiment was repeated three times.

| Cell apoptosis assay
We performed this assay using an Annexin V-Fluorescein Each experiment was repeated three times.

| Statistical analysis
All data are expressed as the mean ± standard deviation (SD) and were analysed using SPSS software version 19.0 (IBM Corp.). We performed statistical analysis using one-way analysis of variance (ANOVA) and Dunnett's post hoc test. For independent two-group analyses, Student's t tests were used.

| Discovery of ALI-associated lncRNAs via microarray analysis
We investigated the differential expression of lncRNAs/mRNAs in alveolar macrophages from lung subsegments instilled with LPS using raw microarray data obtained from the NCBI GEO database (GSE40885). Of the 1289 lncRNAs/mRNAs detected via microarray analysis, 1011 were upregulated in alveolar macrophages from LPS-instilled lung subsegments compared to controls when using the criteria of mean |FC| > 2 and P <.05 ( Figure 1B,C).
Among them, lncRNA HOTAIR as an oncogene has been confirmed by numerous studies and plays a key role in tumour development. 21,22 However, the role and molecular mechanism of lncRNA HOTAIR in ALI have not been reported yet. Therefore, we chose HOTAIR gene as the target lncRNA of this study for further discussion.
In addition, we identified the top 23 pathways associated with these differentially expressed lncRNAs via KEGG pathway analysis.
Of these 23, the most significantly enriched and relevant were the interleukin-18 (IL-18) signalling pathway as well as the senescence and autophagy in cancer signalling pathway, with the latter corresponding to 14 upregulated genes.

| MiR-17-5p inhibition reversed the effects of si-HOTAIR on cell autophagy, proliferation and apoptosis
Using the starBase online database, we found that miR-17-5p was a potential target of HOTAIR ( Figure 4A). Dual LRA results confirmed that miR-17-5p directly interacted with HOTAIR ( Figure 4B). In order to verify the miR-17-5p-HOTAIR interaction, RNA pull-down was employed. The results indicated that HOTAIR and miR-17-5p RNA level were significantly higher in A549 and BEAS-2B cells compared to control and Mut groups ( Figure 4C,D). Furthermore, RT-qPCR results confirmed that LPS reduced miR-17-5p expression and that si-HOTAIR reversed this reduction in A549 and BEAS-2B cells ( Figure 4E).
RT-qPCR further confirmed LRA results ( Figure 4F). FCM indicated that miR-17-5p inhibition increased apoptosis ( Figure 4G,H) and arrested LPS-induced A549 and BEAS-2B cells in the G0/G1 phase prior to their entry into S phase ( Figure 4I,J) after co-transfection with si-HOTAIR compared to the NC inhibitor. Further, the si-HOTAIR-mediated increase in cell proliferation was reversed in the miR-17-5p inhibitor group compared with the NC inhibitor group ( Figure 4K). The protein expression of autophagy markers LC3B and Beclin-1 was increased after co-transfection with si-HOTAIR and miR-17-5p inhibitor compared with the NC inhibitor group ( Figure 5A,B). Similarly, less LPS-induced autophagy vacuoles were observed under NC co-transfection, whereas miR-17-5p inhibitor co-transfection had the opposite effect ( Figure 5C). Altogether, miR-17-5p inhibition counteracted the si-HOTAIR-mediated suppression of autophagy, apoptosis, cell cycle progression and proliferation of A549 and BEAS-2B cells.
Subsequently, WB indicated that their protein expression was enhanced by LPS, whereas si-HOTAIR transfection reversed this effect.

| Si-HOTAIR significantly reversed LPS-induced ALI in vivo
Lung tissue reflects the severity of LPS-induced ALI in mice. After

| D ISCUSS I ON
HOTAIR, a cell cycle-associated lncRNA, is linked to a range of major diseases, including cancer. 23,24 Studies have shown that lncRNA is closely related to cellular functions such as proliferation and apoptosis as well as cancer cell migration and invasion. [25][26][27] However, the underlying mechanisms of HOTAIR in ALI remain poorly understood.
A key aspect of the current study is that we provided a compre-  are associated with ALI. In our study, the upregulation of ATG2, ATG7 and ATG16 occurred in parallel to the inhibition of LPSstimulated A549 and BEAS-2B cell proliferation. These findings highlighted the therapeutic potential of afore-mentioned ATGs as drug targets.
In conclusion, our findings elucidate the molecular mechanisms of HOTAIR underlying ALI. We confirmed the upregulation of HOTAIR in LPS-induced ALI cell models. Further, functional experiments indicated that HOTAIR affected autophagy, apoptosis, the cell cycle and proliferation by regulating miR-17-5p, highlighting the therapeutic relevance of this signalling axis in LPS-induced ALI.

ACK N OWLED G EM ENTS
None.

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.