Yan Li,Chunmei Zhang,Zhongyan Zhao
CircSLCO3A1 depletion ameliorates lipopolysaccharide-induced inflammation and apoptosis of human pulmonary alveolar epithelial cells through the miR-424-5p/HMGB3 pathway
ALI,CircSLCO3A1,miR-424-5p,HMGB3
Background Recent studies have shown the pathogenesis of acute lung injury (ALI) involves circular RNA (circRNA). However, there are no data on the role of circSLCO3A1 in ALI and the underlying mechanism. Objective ALI-like cell injury was induced by stimulating human pulmonary alveolar epithelial cells (HPAEpiCs) using lipopolysaccharide (LPS). The expression of circSLCO3A1, miR-424-5p and high mobility group box 3 (HMGB3) was detected by quantitative real-time polymerase chain reaction. Cell viability and cell apoptosis were assessed by cell counting kit-8 (CCK-8) assay and flow cytometry analysis, respectively. Enzyme-linked immunosorbent assay was performed to determine the production of interleukin-1β (IL-1β), IL-6, tumor necrosis factor-α (TNF-α) and monocyte chemotactic protein 1 (MCP-1). Caspase-3 activity was detected by caspase-3 activity assay. Protein expression of inducible NOS (iNOS), cyclooxygenase-2 (COX2), p-p65 and p65 was analyzed by Western blot. The interactions among circSLCO3A1, miR-424-5p and HMGB3 were identified by dual-luciferase reporter assay, RNA immunoprecipitation assay and RNA pull-down assay. Results CircSLCO3A1 and HMGB3 expression were significantly increased, while miR-424-5p was decreased in LPS-treated HPAEpiCs and the serum of septic ALI patients in comparison with controls. CircSLCO3A1 knockdown assuaged LPS-induced HPAEpiC inflammation and apoptosis. Besides, circSLCO3A1 targeted miR-424-5p and regulated LPS-triggered HPAEpiC inflammation and apoptosis by binding to miR-424-5p. Under the treatment of LPS, miR-424-5p mediated HPAEpiC disorders by targeting HMGB3. Importantly, circSLCO3A1 modulated HMGB3 production by interacting with miR-424-5p. Conclusion CircSLCO3A1 absence assuaged LPS-induced HPAEpiC inflammation and apoptosis through the miR-424-5p/HMGB3 axis. Highlights CircSLCO3A1 expression was upregulated in LPS-induced HPAEpiCs and sepsis-induced ALI patients. CircSLCO3A1 depletion protected against LPS-induced HPAEpiC disorders. CircSLCO3A1 bound to miR-424-5p in HPAEpiCs. MiR-424-5p targeted HMGB3 in HPAEpiCs. CircSLCO3A1 regulated HMGB3 expression through miR-424-5p. Supplementary Information The online version contains supplementary material available at 10.1007/s13273-023-00341-6.
CircSLCO3A1 depletion ameliorates lipopolysaccharide-induced inflammation and apoptosis of human pulmonary alveolar epithelial cells through the miR-424-5p/HMGB3 pathway

Recent studies have shown the pathogenesis of acute lung injury (ALI) involves circular RNA (circRNA). However, there are no data on the role of circSLCO3A1 in ALI and the underlying mechanism.

ALI-like cell injury was induced by stimulating human pulmonary alveolar epithelial cells (HPAEpiCs) using lipopolysaccharide (LPS). The expression of circSLCO3A1, miR-424-5p and high mobility group box 3 (HMGB3) was detected by quantitative real-time polymerase chain reaction. Cell viability and cell apoptosis were assessed by cell counting kit-8 (CCK-8) assay and flow cytometry analysis, respectively. Enzyme-linked immunosorbent assay was performed to determine the production of interleukin-1β (IL-1β), IL-6, tumor necrosis factor-α (TNF-α) and monocyte chemotactic protein 1 (MCP-1). Caspase-3 activity was detected by caspase-3 activity assay. Protein expression of inducible NOS (iNOS), cyclooxygenase-2 (COX2), p-p65 and p65 was analyzed by Western blot. The interactions among circSLCO3A1, miR-424-5p and HMGB3 were identified by dual-luciferase reporter assay, RNA immunoprecipitation assay and RNA pull-down assay.

CircSLCO3A1 and HMGB3 expression were significantly increased, while miR-424-5p was decreased in LPS-treated HPAEpiCs and the serum of septic ALI patients in comparison with controls. CircSLCO3A1 knockdown assuaged LPS-induced HPAEpiC inflammation and apoptosis. Besides, circSLCO3A1 targeted miR-424-5p and regulated LPS-triggered HPAEpiC inflammation and apoptosis by binding to miR-424-5p. Under the treatment of LPS, miR-424-5p mediated HPAEpiC disorders by targeting HMGB3. Importantly, circSLCO3A1 modulated HMGB3 production by interacting with miR-424-5p.

CircSLCO3A1 absence assuaged LPS-induced HPAEpiC inflammation and apoptosis through the miR-424-5p/HMGB3 axis.

CircSLCO3A1 expression was upregulated in LPS-induced HPAEpiCs and sepsis-induced ALI patients. CircSLCO3A1 depletion protected against LPS-induced HPAEpiC disorders. CircSLCO3A1 bound to miR-424-5p in HPAEpiCs. MiR-424-5p targeted HMGB3 in HPAEpiCs. CircSLCO3A1 regulated HMGB3 expression through miR-424-5p.

The online version contains supplementary material available at 10.1007/s13273-023-00341-6.

Acute lung injury (ALI) is an inflammatory lung disease that is induced by bacterial infection with about a 30–50% mortality rate, featured by overwhelming hyperinflammation in the lung (Mowery et al. 2020). The severe form of the inflammatory lung disorder is acute respiratory distress syndrome, which commonly causes fulminant respiratory failure and death (Butt et al. 2016). In spite of the decreased mortality rate of ALI cases after several advanced treatments, ALI remains a major challenge for clinicians (Bellani et al. 2016). The pathogenesis of ALI involves inflammatory responses, the production of inflammatory mediators like IL-1β and IL-6 and alveolar epithelial cell apoptosis (Hendrickson and Matthay 2018; Monsel et al. 2016; Qiu et al. 2020). Thus, an in-depth investigation on the inflammatory and apoptotic pathways may be necessary for developing therapeutic strategies for ALI. Circular RNA (circRNA) is an endogenous transcript that is generated by aberrant back-splicing of pre-mRNA and participates in the pathophysiological process of diseases (Bach et al. 2019). CircRNA is resistant to exonuclease, and the stable structure makes the molecule an ideal candidate for disease treatment (Arnaiz et al. 2019). Considerable work has shown that circRNA regulates cell processes by sponging RNA-binding proteins or microRNA (miRNA), interacting with host genes or functioning as a protein scaffold (Kristensen et al. 2019). Previous studies reported that circRNA regulated the pathogenesis of ALI. Zhu et al. showed that circ_0001679 depletion led to decreases of lipopolysaccharide (LPS)-induced mouse lung epithelial cell apoptosis and pro-inflammatory cytokine production (Zhu et al. 2022). CircRNA paralemmin 2 absence attenuated LPS-induced inflammatory cytokine production as well as oxidative stress, as revealed by ALI mouse model and ALI cell model (Ren et al. 2022). Another circRNA, circSLCO3A1 (hsa_circ_0003252), has been confirmed to be highly expressed in lung tissues from influenza A virus-induced C57B/L6 mice in comparison with control groups (Wang et al. 2021), while whether the occurrence of human ALI is related to circSLCO3A1 remains to be explored. MiRNA has a length of 18–25 nucleotides and can promote mRNA degradation or translation inhibition (Ambros 2004). The expression of miRNAs changes in different diseases like neurodegenerative and inflammatory diseases (Ferruelo et al. 2018). Modification in miRNA expression mediates cell inflammation and tissue repair mechanisms in ALI (Bhargava and Wendt 2012). Recently, Ju and his colleagues found miR-27a could attenuate LPS-caused ALI using LPS-challenged mice (Ju et al. 2018). In addition, the decreased expression of miR-34b-5p attenuated LPS-caused lung apoptosis and inflammation through interaction with progranulin (Xie et al. 2018). MiR-424-5p, a small miRNA, also participates in the pathogenesis of ALI through fibroblast growth factor 2 (FGF2) (Cheng et al. 2020). Through prediction of starbase database, we found circSLCO3A1 possessed the binding sites of miR-424-5p. We intend to analyze whether circSLCO3A1 exerts function by binding to miR-424-5p in ALI. Herein, we stimulated human pulmonary alveolar epithelial cells (HPAEpiCs) using LPS to mimic ALI-like cell injury to analyze the function and the detailed mechanism of circSLCO3A1 in ALI in vitro. In addition, we assessed the association of circSLCO3A1 and miR-424-5p in LPS-caused HPAEpiC disorders. Given that circRNAs could regulate mRNAs in a miRNA-dependent manner (Kristensen et al. 2019), we also assembled the circSLCO3A1/miR-424-5p/mRNA axis to explain the mechanism by which circSLCO3A1 mediated ALI occurrence.

After approval from the Ethics Committee of China-Japan Union Hospital of Jilin University and obtaining the written informed consent from septic ALI sufferers, we collected serum samples from septic ALI sufferers (N = 33) and healthy volunteers (N = 18) in China-Japan Union Hospital of Jilin University. These samples were immediately stored at 4 °C. Pathological examination was carried out by two experienced pathologists to confirm ALI.

HPAEpiCs were purchased from Cell Research (Shanghai, China) and maintained in alveolar epithelial cell medium (AEpiCM; Cell Research) added with penicillin&streptomycin solution (Cell Research) and 10% fetal bovine serum (Cell Research) at 37 °C with 5% CO2. The construction of ALI cell model was performed by inducing HPAEpiCs with 1 μg/mL LPS (Sigma, Louis, MO, USA) for 24 h according to the results of the previous study (Nan et al. 2020).

GenePharma (Shanghai, China) provided the small interfering RNA against circSLCO3A1 (si-circSLCO3A1, 5ʹ-CTATATAGGTGAGCGTCCTGA-3ʹ), miR-424-5p mimics (miR-424-5p, 5ʹ-CAGCAGCAAUUCAUGUUUUGAA-3ʹ), miR-424-5p inhibitors (anti-miR-424-5p, 5ʹ-UUCAAAAGAUGAAUUGCUGCUG-3ʹ) and the respective controls (si-NC, miR-NC and anti-NC). The overexpression plasmid of high mobility group box 3 (HMGB3) was achieved by inserting its 603 bp coding fragment into the pcDNA 3.1(+) vector, and named pcDNA 3.1-HMGB3. Transfection (20 pmoL of si-circSLCO3A1, 60 nM of miR-424-5p, 100 nM of anti-miR-424-5p, 20 pmoL of si-NC, 60 nM of miR-NC, 100 nM of anti-NC, and 0.8 μg of pcDNA 3.1-HMGB3) was performed on HPAEpiCs cultured in 24-well plates (at about 70% of fusion rate) at the same time using FuGENE6 (Roche, Basel, Switzerland) as per the established guidelines, and cells were then treated with 1 μg/mL LPS (Sigma) for 24 h to induce injury.

Trizol (TaKaRa, Dalian, China) method was conducted for isolating RNA from serum samples and HPAEpiCs. Then, preparation of circSLCO3A1 and HMGB3 cDNA was performed using FastKing RT kits (Tiangen, Beijing, China), while miR-424-5p first stand cDNA was synthesized using miRNA synthesis reagents (TaKaRa). The resultant cDNAs were quantified using ABI StepOne qRT-PCR System with SYBR® Premix (TaKaRa) and primers (shown in Table 1). Results were determined with the 2−∆∆Ct method with normalization to GAPDH or U6.

Construction of circSLCO3A1 was analyzed using convergent primer, divergent primer and RNase R. In brief, cDNA and gDNA were obtained from HPAEpiCs using a DNA synthesis kit (TaKaRa), and then qRT-PCR was performed with convergent and divergent primers. In addition, RNA from HPAEpiCs was incubated with RNase R (Tiangen) for 30 min in an incubator. At last, circSLCO3A1 and SLCO3A1 expression were analyzed to confirm circSLCO3A1 construction.

Nucleocytoplasmic separation of HPAEpiCs was carried out with a subcellular fractionation location kit (Amyjet, Wuhan, China) based on the established guidelines. In brief, HPAEpiCs were incubated with CEB-A reagent and then homogenized in a tissue homogenizer, followed by 2-min centrifugation at 500 g. The supernatant was transferred into tubes. Finally, nucleocytoplasmic RNA was subjected to qRT-PCR analysis.

HPAEpiCs cultured in 96-well petri dishes were treated with 1 μg/mL LPS (Sigma), si-circSLCO3A1, anti-miR-424-5p, pcDNA3.1-HMGB3, si-NC, anti-NC, or pcDNA3.1 vector alone or jointly. After 48 h of transfection, the cells were incubated with CCK-8 reagent (Dojindo, Shanghai, China), lasting 3 h. Absorbance at 450 nm was analyzed using an enzyme immunoassay analyzer (REAGEN, Shenzhen, China).

Commercial ELISA kits (Beyotime, Shanghai, China) were used to analyze the levels of IL-1β, IL-6, tumor necrosis factor-α (TNF-α) and monocyte chemotactic protein 1 (MCP-1) as per the guidebooks. In brief, HPAEpiC supernatant was harvested and incubated with Biotin-Conjugate antibodies as well as Streptavidin. Eventually, a microplate reader (REAGEN) was applied to examine the samples.

Ice-cold lysis buffer (Beyotime) was utilized to lyse HPAEpiCs. The proteins were separated and transferred onto polyvinylidene fluoride membrane, which was blocked in nonfat dried milk. Subsequently, the polyvinylidene fluoride membranes were incubated with the following primary antibodies: anti-inducible NOS (anti-iNOS; Cat#MA5-17139; 1:1000; Thermo Fisher, Waltham, MA, USA), anti-cyclooxygenase-2 (anti-COX2; Cat#MA5-14568; 1:1000; Thermo Fisher), anti-p-p65 (Cat#AF2006; 1:2000; Affinity, Nanjing, China), anti-p65 (Cat#AF5006; 1:1000; Affinity) and anti-GAPDH (Cat#MA1-16757; 1:1000; Thermo Fisher). After being washed with PBS, secondary antibodies (Cat#A32731/A32723) were used to incubate the membranes. At last, immunoreactive bands were visualized using eyoECL Plus (Beyotime).

HPAEpiCs were seeded in 12-well plates with a density of 1 × 105 cells per well. All cells were collected after various treatments and then suspended in binding buffer. The cells were subjected to flow cytometry analysis after Annexin V-FITC (Solarbio, Beijing, China) and propidium iodide (Solarbio) double staining in the dark.

The assay regarding the detection of caspase3 activity was carried out based on the instruction of Caspase-3 Activity Assay Kit (Abcam, Cambridge, MA, USA). HPAEpiCs were seeded in 96-well plates and cultured overnight in AEpiCM plus 10% fetal bovine serum. Then, the cells were subjected to incubation with phenol-red free media (PRFM), working-concentration Staurosporine and cold Caspase Cell Lysis Buffer. At last, a fluorescent microplate reader was used to measure fluorescence.

Online database starbase (http://starbase.sysu.edu.cn/agoClipRNA.php?source=mRNA) was used to search for the target miRNA of circSLCO3A1 and the target gene of miR-424-5p. The wild-type reporter plasmids of circSLCO3A1 (WT-circ_circSLCO3A1) and HMGB3 3’-untranslated region (3’UTR) (WT-HMGB3 3’UTR) were achieved by inserting their linear sequences containing miR-424-5p-binding sites into the pmirGLO vector (YouBio, Changsha, China). In addition, the sequences of circSLCO3A1 and HMGB3 3’UTR with site-directed mutagenesis were used to generate mutant plasmids, including MUT-HMGB3 3’UTR and MUT-HMGB3 3’UTR. Subsequently, the reporter plasmids were transfected into HPAEpiCs with miR-424-5p mimics or miR-NC for 48 h before detecting luciferase activity using Dual-Lucy Assay Kit (Solarbio).

The ago2-RIP experiment was conducted in HPAEpiCs. After 48 h of culture, HPAEpiCs were dissolved in RIP Lysis Buffer. According to the guidebook of Magna RIP kit (Millipore, Billerica, MA, USA), the lysates were incubated with magnetic beads conjugated with anti-ago2 antibody or IgG antibody (Abcam) for 24 h. The RNA complexes were subjected to qRT-PCR.

MiR-424-5p and miR-NC were biotinylated by Sangon (Shanghai, China) to generate bio-miR-424-5p and bio-NC. Subsequently, HPAEpiCs were transfected with these biotinylated oligonucleotides. Forty-eight hours later, the cells were collected and exposed to RIP lysis buffer, followed by incubation with streptavidin-coupled beads (Sigma), lasting 4 h. At last, the biotin-coupled RNA complexes were analyzed by qRT-PCR.

Data from three independent and not replicate experiments were analyzed by GraphPad Prism. Results were expressed as means ± standard deviations. Significant differences were compared with two-tailed Student’s t tests or Wilcoxon-Mann–Whitney test between the two groups, while with one-way analysis of variance among three or more groups. P < 0.05 indicated statistical significance.

CircSLCO3A1 (also named circ_0003252) was located on the chr15:92459222–92459688 and formed by the cyclization of exon 2 of the SLCO3A1 gene with 466 bp in size (Fig. 1A). We confirmed its circular construction in HPAEpiCs using convergent and divergent primers. For instance, circSLCO3A1 was detected only from gDNA, while liner SLCO3A1 was detected from both cDNA and gDNA (Fig. 1B). Moreover, circSLCO3A1 was tolerant to RNase R digestion in comparison with the linear gene SLCO3A1 (Fig. 1C). In addition, we found that circSLCO3A1 was mainly expressed in the cytoplasm of HPAEpiCs (Fig. 1D). Further, we observed that circSLCO3A1 expression was significantly upregulated in LPS-induced HPAEpiCs (Fig. 1E). Thus, these results showed that circSLCO3A1 had a circular construction and was highly expressed in LPS-induced HPAEpiCs.

Then, we analyzed the functional effects of circSLCO3A1 in LPS-induced HPAEpiCs. The results of Fig. 2A showed that circSLCO3A1 expression was dramatically reduced after transfection with si-circSLCO3A1 compared with the control group, showing the success of circSLCO3A1 knockdown. Subsequently, the results showed that LPS treatment increased circSLCO3A1 expression, while the effect was remitted when circSLCO3A1 expression was downregulated (Fig. 2B). As presented in Fig. 2C, LPS exposure led to decreased cell viability, but the effect was restored after circSLCO3A1 depletion. In addition, we observed that LPS treatment increased the production of inflammation-related IL-1β, IL-6, TNF-α and MCP-1 in HPAEpiCs; however, these effects were attenuated by decreasing circSLCO3A1 expression (Fig. 2D and E). We also detected the expression of inflammation-related proteins (iNOS, COX2, p-p65 and p65) by Western blot in the cells. As expected, LPS treatment promoted iNOS and COX2 production and increased the value of p-p65/p65, but these impacts were counteracted after transfection with si-circSLCO3A1 (Fig. 2F–I). Further, LPS treatment-induced increases of HPAEpiC apoptosis and caspase3 activity were relieved by reducing circSLCO3A1 expression (Fig. 2J and K). Collectively, the above evidence suggested that circSLCO3A1 was involved in LPS-induced inflammation and apoptosis of HPAEpiCs.

Starbase database was performed to predict the target miRNA of circSLCO3A1. As shown in Fig. 3A, miR-424-5p contained the complementary sites of circSLCO3A1. Then, we carried out dual-luciferase reporter assay, RIP assay and RNA pull-down assay to identify the association of circSLCO3A1 and miR-425-5p. The success of miR-424-5p overexpression in HPAEpiCs was presented in Fig. 3B. As revealed in Fig. 3C, miR-424-5p dramatically inhibited the luciferase activity of wild-type reporter plasmid of circSLCO3A1 compared with the controls. Moreover, the results showed that both circSLCO3A1 and miR-424-5p were greatly enriched by the ago2 antibody compared with the IgG antibody (Fig. 3D). RNA pull-down assay showed that the expression of circSLCO3A1 was higher in the bio-miR-424-5p group than in the bio-NC group (Fig. 3E). In addition, qRT-PCR analysis revealed that circSLCO3A1 depletion increased miR-424-5p expression in HPAEpiCs (Fig. 3F). Further, we observed that LPS stimulation decreased miR-424-5p expression in HPAEpiCs (Fig. 3G). These results demonstrated that circSLCO3A1 directly bound to miR-424-5p in HPAEpiCs.

The study silenced both circSLCO3A1 and miR-424-5p in LPS-induced HPAEpiCs to determine the consequent effects on cell inflammation and apoptosis. The data from Fig. 4A first showed the success of miR-424-5p knockdown in HPAEpiCs. Subsequently, the expression analysis of circSLCO3A1 in the various groups was shown in Figure S1A. The results showed that circSLCO3A1 absence attenuated the inhibitory effects of LPS treatment on miR-424-5p expression and cell viability, but these results were remitted after transfection with anti-miR-424-5p (Fig. 4B and C). LPS-induced dysregulation of IL-1β, IL-6, TNF-α, MCP-1, iNOS, COX2 and p-p65/p65 was remitted by decreasing circSLCO3A1 expression, while these effects were restored after miR-424-5p knockdown (Fig. 4D–I). Consistently, the increased cell apoptosis and caspase3 activity by LPS were relieved after circSLCO3A1 depletion; however, these effects were attenuated by reducing miR-424-5p expression (Fig. 4J–L). Together, these findings manifested that the circSLCO3A1/miR-424-5p regulated LPS-induced HPAEpiC disorders.

The target gene of miR-424-5p was further predicted through starbase online database. As presented in Fig. 5A, miR-424-5p potentially bound to HMGB3. Subsequently, the results showed that miR-424-5p introduction dramatically repressed the luciferase activity of WT-HMGB3 3’UTR but not that of controls (vector or MUT-HMGB3 3’UTR) (Fig. 5B). Also, we found a higher expression of HMGB3 and miR-424-5p in the ago2-induced silencing complex compared with the IgG-induced silencing complex (Fig. 5C). In addition, it was revealed that HMGB3 could be significantly pulled down by bio-miR-424-5p compared to that by bio-NC (Fig. 5D). As expected, HMGB3 expression was downregulated by increasing miR-424-5p expression but upregulated by decreasing miR-424-5p expression (Fig. 5E). Comparatively, circSLCO3A1 depletion inhibited HMGB3 production, which was attenuated after miR-424-5p knockdown (Fig. 5F). Further, the results showed that LPS stimulation induced HMGB3 production in HPAEpiCs (Fig. 5G). Thus, all data demonstrated that circSLCO3A1 regulated HMGB3 expression through miR-424-5p in HPAEpiCs.

Given the regulation of circSLCO3A1 on HMGB3 expression, we continued to determine whether HMGB3 participated in circSLCO3A1 knockdown-mediated actions in HPAEpiCs after LPS treatment. The data first showed the success of HMGB3 overexpression (Fig. 6A). The expression analysis of circSLCO3A1 in the various groups was shown in Figure S1B. Subsequently, circSLCO3A1 depletion reduced HMGB3 production in LPS-treated HPAEpiCs, whereas the effect was remitted when HMGB3 expression was increased (Fig. 6B). Moreover, circSLCO3A1 absence increased cell viability and decreased IL-1β, IL-6, TNF-α and MCP-1 production in LPS-treated HPAEpiCs, but these effects were rescued by increasing HMGB3 expression (Fig. 6C–E). Consistently, ectopic HMGB3 expression also remitted the inhibitory effects of circSLCO3A1 depletion on iNOS and COX2 production and the value of p-p65/p65 in LPS-stimulated HPAEpiCs (Fig. 6F–I). Further, the decreased cell apoptosis and caspase3 activity by si-circSLCO3A1 were remitted after transfection with pcDNA3.1-HMGB3 in LPS-treated HPAEpiCs (Fig. 6J and K). Collectively, all results suggested that circSLCO3A1 mediated LPS-induced HPAEpiC inflammation and apoptosis through HMGB3.

Further, we analyzed circSLCO3A1, miR-424-5p and HMGB3 expression in the serum of septic ALI patients through qRT-PCR. As shown in Fig. 7A–C, circSLCO3A1 and HMGB3 expression were significantly upregulated, while miR-424-5p was downregulated in the serum of septic ALI patients. Moreover, we found that circSLCO3A1 expression was negatively correlated with miR-424-5p expression but positively with HMGB3 expression in the serum of septic ALI patients (Fig. 7D and E). The data from Fig. 7F also showed the negative correlation of miR-424-5p and HMGB3 expression in the serum of septic ALI patients.

LPS is an important medium for sepsis and can activate LPS-binding protein complex of CD14/TLR4 receptor on some types of cells, such as monocytes, and macrophages, thereby inducing the production of inflammatory mediators (Reutershan et al. 2005). On that account, LPS has been widely utilized to mimic ALI models (Li et al. 2016a, 2021). In the present work, using a LPS-induced ALI cell model, we found the beneficial effects of circSLCO3A1 depletion against LPS-induced human pulmonary alveolar epithelial cell injuries for the first time, which was attributed to its anti-apoptotic and anti-inflammatory properties. Subsequent functional assays explained that circSLCO3A1 regulated human pulmonary alveolar epithelial cell dysfunction through miR-424-5p/HMGB3 pathway. As reported, circRNA is generated from precursor mRNA and is classified into exonic circRNA, intronic circRNA as well as exon–intron circRNA. Exonic circRNAs are transferred to cytoplasm (Lei et al. 2020). CircSLCO3A1 was formed by back-splicing of the exon 2 of the SLCO3A1 gene, and we could detect its expression in the cytoplasm of HPAEpiCs. Moreover, we found that circSLCO3A1 was highly expressed in LPS-treated HPAEpiCs and septic ALI patients. More and more studies explain that pro-inflammatory cytokine production, a key event in ALI, is related to clinical outcome of patients with lung injury (Gong et al. 2017; Li et al. 2016b). In this study, we observed that LPS stimulation induced HPAEpiC inflammation by increasing the secretion of pro-inflammatory cytokines (IL-1β, IL-6, TNF-α and MCP-1), the production of iNOS and COX2 and the phosphorylation of p65. In addition, LPS treatment increased cell apoptotic rate and caspase3 activity. Loss-of-function assays showed that circSLCO3A1 depletion attenuated LPS-induced cell apoptosis and inflammation, suggesting that circSLCO3A1 overexpression might contribute to ALI occurrence. CircRNAs can negatively modulate miRNA expression by serving as competing endogenous RNAs (Panda 2018). Herein, we identified circSLCO3A1 targeted and negatively modulated miR-424-5p in HPAEpiCs. As reported, miR-424-5p participated in the occurrence of inflammation-related diseases, such as atherosclerosis (Li et al. 2020), endometritis (Umar et al. 2022) and acute kidney injury (Fan et al. 2021). In addition, miR-424-5p could ameliorate sepsis-caused ALI by reducing the number of inflammatory cells and repressing mice apoptosis through Rho-associated kinase 2 (ROCK2) (Chen et al. 2020). Another reference documented that miR-424-5p combined with FGF2 to repress LPS-caused alveolar epithelial cell apoptosis and inflammation (Cheng et al. 2020). Consistently, our data also indicated the beneficial effect of miR-424-5p against LPS-induced cell damage. Meanwhile, miR-424-5p was downregulated in LPS-stimulated HPAEpiCs and sepsis-induced ALI patients in comparison with control groups. Importantly, our findings demonstrated that circSLCO3A1 depletion ameliorated LPS-induced cell injuries owing to miR-424-5p overexpression. HMGB3, also termed HMG2a, is an abundant nonhistone protein that belongs to a member of the HMG super family, participating in DNA replication and the formation of nucleoprotein complex (Nemeth et al. 2003). The protein is associated with DNA-binding proteins to regulate transcription. A previous study demonstrated that HMGB3 was expressed in tumor tissues and was responsible for cancer recurrence and drug resistance (Wen et al. 2021). Additionally, HMGB3 was positively expressed in serum of ARDS/ALI patients (Ueno et al. 2004). Our data confirmed miR-424-5p bound to HMGB3 in HPAEpiCs and that HMGB3 was upregulated in LPS-stimulated HPAEpiCs and sepsis-induced ALI patients. Moreover, HMGB3 expression was positively correlated with circSLCO3A1 but negatively with miR-424-5p. Rescue assays demonstrated that circSLCO3A1 mediated HMGB3 expression through miR-424-5p. A recent investigation indicated that HMGB3 could aggravate LPS-induced lung epithelial apoptosis as well as inflammation (Sun et al. 2021). Similarly, the present work also found the promoting effect of HMGB3 on LPS-induced cell injuries. Further, functional assays manifested that HMGB3 was responsible for the regulation of circSLCO3A1 toward LPS-caused cell damage. Taken together, we confirmed the high expression of circSLCO3A1 in lipopolysaccharide-induced human pulmonary alveolar epithelial cells and ALI patients. The study innovatively put forward and confirmed circSLCO3A1 depletion repressed LPS-caused cell apoptosis and inflammation by decreasing pro-inflammatory cytokine production and inhibiting cell apoptosis via combining with miR-424-5p/HMGB3 axis in vitro (Fig. 8). Nevertheless, a mouse model assay should be performed to validate the in vitro data about the function of circSLCO3A1 in ALI and the underlying mechanism. Even so, these results may provide a theoretical direction for acute lung injury.

Below is the link to the electronic supplementary material.Supplementary file1 The expression of circSLCO3A1 was analyzed by qRT-PCR. HPAEpiCs were divided into control group (untreated cells), LPS group, LPS+si-circSLCO3A1 group or LPS+si-circSLCO3A1+anti-miR-424-5p group, and circSLCO3A1 expression was analyzed by qRT-PCR (A). HPAEpiCs were divided into control group (untreated cells), LPS group, LPS+si-circSLCO3A1 group or LPS+si-circSLCO3A1+pcDNA3.1-HMGB3 group, and circSLCO3A1 expression was analyzed by qRT-PCR (B). Three independent experiments with biological repeats. ***P < 0.001 (TIF 218 KB)