Has_circRNA_0122683 (circ-PRKCI) relieves ferroptosis of HPAEpiCs in sepsis-induced acute lung injury by sponging miR-382-5p

Plasma samples

This study was a retrospective research, the protocols and procedures of clinical samples related experiments were strictly reviewed and permitted by the Ethics Committee of The People’s Hospital of Suzhou New District following the Helsinki Declaration (No. 2024-110), and written informed consent was obtained from all participants or their delegates before the beginning of this study. The venous blood samples (5 mL) were collected from 80 sepsis patients (included 36 women and 44 men with an average age of 46.17 years) and 53 age and gender-matched healthy individuals (included 21 women and 32 men with an average age of 44.90 years) recruited between May 2022 and May 2024 from The People’s Hospital of Suzhou New District (Suzhou, China). The 80 sepsis patients all had bacterial infections, including 31 septic ALI patients and 49 septic non-ALI patients. Patients who transferred from other hospitals, received immunosuppressive therapy, pregnant or lactating were excluded. Sepsis and sepsis-induced ALI were diagnosed according to the corresponding international criteria (Singer et al., 2016; Raghavendran & Napolitano, 2011). Healthy individuals had no history of sepsis or other severe infection or systemic diseases. All plasma samples were obtained from peripheral venous blood by centrifugation at 3,000× g for 10 min at 37 °C, and were immediately frozen in liquid nitrogen and subsequently stored at −80 °C until RNA extraction.

Cell culture and LPS exposure

Human pulmonary alveolar epithelial cells (HPAEpiCs) were acquired from Shanghai Honsun Biological Technology Co., Ltd. (Shanghai, Beijing, China), and maintained in Dulbecco’s Modified Eagle Medium (DMEM; Life Technologies, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS; Gibco, Waltham, MA, USA) and 1% penicillin/streptomycin (Sigma-Aldrich, St. Louis, MO, USA). The cell culture condition was 5% CO₂ and 95% air at a constant temperature of 37 °C. To construct sepsis-ALI cell model, HPAEpiCs were treated with 1 μg/mL of LPS (Beyotime Biotechnology, Shanghai, China) for different times (3, 6, 12, or 24 h), as previously described (Zhang et al., 2021a).

Cell transfection

To overexpress circ-PRKCI or miR-382-5p, circ-PRKCI overexpression vector (circ-PRKCI) and the corresponding control vector (circ-NC), miR-382-5p mimic (5′-GAAGUUGUUCGUGGUGGAUUCG-3′) and mimic negative control (mimic NC; 5′-GGUUCGUACGUACACUGUUCA-3′), were synthesized from Guangzhou Ribobio Co., Ltd. (Guangzhou, China). When cell confluence reached ~75%, HPAEpiCs were transfected with the above plasmids or oligonucleotides using Lipofectamine 3000 reagent (Invitrogen, Waltham, MA, USA) in accordance with the manufacturer’s manual. A total of 48 h after transfection, the efficiency of the transfection was verified by quantitative real-time PCR (qRT-PCR). After the treatment with 5.0 mmol/L of Erastin or 4.0 μmol/L of FIN56 (ferroptosis inducer; Selleck Chemicals, Houston, TX, USA), the transfected HPAEpiCs were exposed to 1 μg/mL of LPS for further functional experiments.

RNA extraction and qRT-PCR

Total RNA was extracted from plasma samples and cells using TRIZOL reagent (TaKaRa, Kusatsu, Japan), and then quantified by NanoDrop 2000/2000c (Thermo Scientific, Waltham, MA, USA). OD260/OD230 ratios >1.8 but <2.1 were accepted. For synthesized complementary DNA (cDNA) of miRNAs, the RNA was reversely transcribed with the miScript II RT Kit (QIAGEN) according to the manufacturer’s protocols. For cDNA of circRNAs and mRNAs, the RNA was reversely transcribed with using the PrimeScript RT Reagent Kit (TaKaRa, Kusatsu, Japan) in accordance with the manufacturer’s manual. qRT-PCR reaction was performed with the miScript SYBR Green PCR Kit (QIAGEN) or SYBR® Premix Ex Taq™ II reagent kit (TaKaRa, Kusatsu, Japan) on an ABI 7900HT Real-Time PCR System (Applied Biosystems, Waltham, MA, USA) according to the manufacturer’s instructions, with amplification condition at 95 °C for 15 min followed by 40 cycles of 95 °C for 10 s and 59 °C for 30 s and 45 s at 72 °C. The primers used in this study were synthesized from Shanghai Sangon Biotech Co., Ltd (Shanghai, Beijing, China), and the specific sequences were shown in Table S1. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the standardized internal reference of circ-PRKCI and mRNAs, and U6 was utilized as the standardized internal control of miRNAs, and relative expression level of each gene was calculated using the 2^−ΔΔCt method.

Ribonuclease R (RNase R) and Actinomycin D (ActD) treatment

For Ribonuclease R (RNase R) treatment, 3 μg of total RNA from HPAEpiCs was treated with 2 U/μg RNase R (Epicenter Biotechnologies) at 37 °C for 15 min to remove the linear RNA. For Actinomycin D (ActD) treatment, when cell confluence reached ~75%, HPAEpiCs were treated with 2.0 μg/mL ActD (MedChem Express, Monmouth Junction, NJ, USA) and collected at different times (0, 4, 8, 12, or 24 h). Afterwards, the RNA was collected, and stability of circ-PRKCI and PRKCI mRNA was evaluated by qRT-PCR.

Luciferase reporter assay

The sequences of circ-PRKCI were obtained from circBase (http://www.circbase.org), and miR-382-5p sequences were derived from miRBase (https://www.mirbase.org/). We predicted the miRNA binding sites of circ-PRKCI using the bioinformatics databases CircInteractome (https://circinteractome.nia.nih.gov/index.html) and circBank1.0 (http://www.circbank.cn/index.html). The luciferase reporter assay was employed to confirm the target binding sites between miR-382-5p and circ-PRKCI. The wild-type (wt) circ-PRKCI sequence or mutant (mut) sequence was inserted into the 3′-UTR downstream region of Renilla luciferase in the psiCHECKTM-2 vector (Promega) to construct corresponding luciferase reporter plasmids. When cell confluence reached ~75%, HPAEpiCs were co-transfected with miR-382-5p mimic or mimic NC and either wt-circ-PRKCI plasmid or mut-circ-PRKCI plasmid or basic plasmid with Lipofectamine 3000 reagent. A luciferase reporter assay kit (Promega) was used to determine the Renilla and Firefly luciferase activity. The relative luciferase activity was shown as Renilla luciferase activity normalized to that of Firefly.

RNA immunoprecipitation (RIP) assay

The Magna RIP Kit (Millipore) was applied for RNA immunoprecipitation (RIP) assay according to the instruction provided by the manufacturer. In brief, magnetic beads were incubated with argonaute2 (Ago2) antibody (Millipore) or corresponding immunoglobulin G (IgG) antibody (Millipore) overnight at 4 °C. Then the beads were incubated with cell lysates from HPAEpiCs, and treated with proteinase K to digest proteins. After the elution of precipitated complex from beads, the immunoprecipitated RNAs were isolated and purified for qRT-PCR analysis. Relative enrichment of miR-382-5p and circ-PRKCI was computed with the normalization to the input.

Detection of cell viability

The viability of cells was examined by Cell Counting Kit-8 (CCK-8) assay according to the manufacturer’s instructions. HPAEpiCs underwent various treatments were seeded in a 96-well plate at a density of 5,000 cells per well and incubated for 24 h. Then, 10 μL of CCK-8 reagent (Beyotime Biotechnology, Shanghai, China) was added into cell culture medium on the indicated time and incubated at 37 °C for 3 h. Finally, cell viability ability was assessed by measuring the absorbance at wavelength of 450 nm using an ultraviolet spectrophotometer (Thermo Scientific, Waltham, MA, USA).

Assessment of pro-infammatory cytokines

The production of pro-infammatory cytokines, including interleukin 6 (IL-6), interleukin 1 beta (IL-1β) and tumor necrosis factor alpha (TNF-α), was measured using enzyme-linked immunosorbent assay (ELISA, Helsinki, Finland). The cell supernatants from HPAEpiCs that underwent various treatments were collected and tested with the corresponding ELISA kits (R&D Systems, Minneapolis, MN, USA) for IL-6, IL-1β and TNF-α according to the manufacturer instructions.

Measurement of iron content (Fe²⁺)

The Fe²⁺ level was measured by the Iron Assay Kit (Abcam). Following the guidelines, HPAEpiCs that underwent various treatments were plated in six-well plates at a density of 3 × 10⁶ cells per well for 24 h. After that, the cells were lysed and cell supernatants were collected by centrifugation at 13,000× g for 15 min at 4 °C. The supernatants were treated with an iron reducer for 30 min at 37 °C, followed by incubation with iron probe for 60 min at 37 °C in the dark. Finally, absorbance was immediately measured with an ultraviolet spectrophotometer at a wavelength of 593 nm.

Glutathione (GSH) assay

The glutathione (GSH) level was detected using the GSH detection kit (Beyotime Biotechnology) according to the manufacturer’s instructions. Briefly, HPAEpiCs that underwent various treatments were seeded into 24-well at density of 1 × 10⁵ cells per well for 24 h. After extraction and centrifugation, the release of GSH in the supernatants was detected using an ultraviolet spectrophotometer at wavelength of 412 nm.

Malondialdehyde (MDA) assay

The malondialdehyde (MDA) concentration was evaluated by the Lipid Peroxidation MDA Detection Kit (Abcam) according to the manufacturer’s protocols. Briefly, HPAEpiCs that underwent various treatments were cultured for 24 h and treated with MDA lysis buffer. Afterward, the cell supernatants were mixed with thiobarbituric acid (TBA) to form an MDA-TBA adduct. With the aid of an ultraviolet spectrophotometer, the absorbance of the MDA-TBA adduct was detected at a wavelength of 532 nm.

Reactive oxygen species (ROS) assay

The Cellular ROS Assay Kit with by H2DCFH-DA fluorescent probe (Abcam, Cambridge, UK) was applied for reactive oxygen species (ROS) detection. HPAEpiCs that underwent various treatments were plated in six-well plates at density of 3 × 10⁶ cells per well for 24 h, and incubated with 10 µmol/L of H2DCFH-DA for 30 min at 37 °C, away from light. After washing with phosphate buffered saline (PBS), images were observed and photographed under a fluorescence microscope (Leica, Wetzlar, Germany), and fluorescence strength was computed with Image J software (National Institutes of Health, Bethesda, MD, USA). All experimental procedures were carried out in strict accordance with the procedures specified in the instructions.

Circ-PRKCI is notably diminished in sepsis-induced ALI

First, to validate the expression pattern of circ-PRKCI in sepsis-induced ALI, we examined the circ-PRKCI expression in plasma samples from 31 septic ALI patients and 49 septic non-ALI patients, as well as 53 healthy individuals. As shown in Fig. 1A, circ-PRKCI was lowly expressed in septic ALI patients compared with healthy controls. Meanwhile, the expression of circ-PRKCI was lower in septic ALI patients than that in septic non-ALI patients. Subsequently, HPAEpiCs were exposed to 1 μg/mL LPS for different times, we found that circ-PRKCI expression was significantly reduced in LPS-treated cells compared to untreated cells, and circ-PRKCI was progressively reduced in a time-dependent manner (Fig. 1B). In addition, we evaluated the stability of circ-PRKCI in HPAEpiCs. After treatment with RNase R, circ-PRKCI was more stable than the linear PRKCI mRNA (Fig. 1C). Also, HPAEpiCs treatment with ActD, which can block new transcription, revealed that circ-PRKCI was more stable in comparison to PRKCI mRNA (Fig. 1D). These results collectively demonstrated that circ-PRKCI is a stable circRNA generated from PRKCI, and is substantially downregulated in sepsis-induced ALI.

The diagnostic performance of circ-PRKCI in sepsis-induced ALI

The potential diagnostic value of circ-PRKCI in sepsis-induced ALI was evaluated through ROC curves, as shown in Fig. 2A, the AUC value for circ-PRKCI in differentiating septic ALI patients from healthy individuals was 0.996 (95% confidence interval (CI) [0.987–1.000]), and the sensitivity and specificity were 96.77% and 100.00%. What is more, circ-PRKCI could discriminate septic ALI patients from septic non-ALI patients with an AUC value of 0.999 (95% CI [0.997–1.000]), sensitivity was 100.00%, and specificity was 97.96% (Fig. 2B). These results indicated that circ-PRKCI may serve as a promising biomarker for clinical diagnosis of sepsis-induced ALI.

Circ-PRKCI ameliorates LPS-induced ferroptosis and inflammatory response of HPAEpiCs

In order to study the possible function of circ-PRKCI on sepsis-induced ALI, we applied circ-PRKCI overexpression vector to enhance endogenous expression of circ-PRKCI in HPAEpiCs. The efficiency for circ-PRKCI overexpression was validated by qRT-PCR, and circ-PRKCI overexpression vector had no significant effects on PRKCI mRNA expression compared with the circ-NC group (Fig. 3A).

Firstly, CCK-8 assay showed that LPS treatment led to a significant decrease in the cell viability of HPAEpiCs, while overexpression of circ-PRKCI could reverse the LPS-induced the inhibition of cell viability (Fig. 3B). Then the levels of Fe²⁺, GSH, MDA, and ROS were detected using corresponding commercial kits. Results revealed that LPS stimulation increased the levels of Fe²⁺_, ROS and MDA in HPAEpiCs, but introduction of circ-PRKCI significantly reduced Fe²⁺, ROS and MDA production (Figs. 3C–3F). Meanwhile, LPS exposure resulted in an inhibitory effect on GSH level, while the effect was reversed following the upregulation of circ-PRKCI (Fig. 3G). Moreover, the ELISA assay showed that LPS administration significantly increased the release of pro-inflammatory factors IL-6, IL-1β and TNF-α in HPAEpiCs, which was attenuated due to circ-PRKCI overexpression (Fig. 4). Additionally, the inhibitory effects of circ-PRKCI overexpression on ferroptosis and inflammatory response in LPS-induced HPAEpiCs were compromised after Erastin or FIN56 treatment. Therefore, we concluded that circ-PRKCI mitigates LPS-induced ferroptosis and inflammatory response of HPAEpiCs, suggesting that circ-PRKCI might play a protective role in sepsis-induced ALI.

miR-382-5p is strikingly upregulated in sepsis-induced ALI and negative correlates with circ-PRKCI expression

To identify miRNAs regulated by circ-PRKCI, we performed bioinformatics analysis using CircInteractome and circBank1.0, and screened three candidate miRNAs including miR-1324, miR-382-5p, and miR-890 (Fig. 5A). Among these miRNAs, only miR-382-5p expression was significantly inhibited in HPAEpiCs by the overexpression of circ-PRKCI (Fig. S1). Moreover, qRT-PCR found that miR-382-5p levels were drastically upregulated in septic ALI patients, relative to healthy individuals and septic non-ALI patients (Fig. 5B). Similarly, miR-382-5p was highly expressed in LPS-induced HPAEpiCs (Fig. 5C). These results suggested a possible regulatory relationship between circ-PRKCI and miR-382-5p.

Circ-PRKCI serves as a ceRNA of miR-382-5p in HPAEpiCs

Circ-PRKCI sequences were noticed to contain the binding sites for miR-382-5p (Fig. 6A). Next, we conducted luciferase reporter assay with wt-circ-PRKCI plasmid or mut-circ-PRKCI plasmid and miR-382-5p mimic co-transfecting into HPAEpiCs. The efficiency of miR-382-5p mimic was validated in HPAEpiCs (Fig. 6B). As shown in Fig. 6C, miR-382-5p mimic significantly reduced the relative luciferase activity of wt-circ-PRKCI plasmid, but failed to reduce the relative luciferase activity of mut-circ-PRKCI plasmid, indicating that miR-382-5p could directly bind to circ-PRKCI. As expected, RIP analysis confirmed that circ-PRKCI and miR-382-5p were significantly enriched in the Ago2 antibody group, compared to the IgG antibody group (Fig. 6D). These results evidenced that circ-PRKCI functions as a sponge for miR-382-5p in HPAEpiCs.

miR-382-5p abolishes the protective effect of circ-PRKCI on LPS-induced HPAEpiCs

Furthermore, to confirm whether the protective effect of circ-PRKCI on LPS-induced HPAEpiCs is targeted by miR-382-5p rescue experiments were performed with a circ-PRKCI overexpression vector together with a miR-382-5p mimic or mimic NC co-transfecting into HPAEpiCs and subsequently subjected to LPS treatment for 24 h. Interestingly, the CCK-8 assay exhibited that overexpression of circ-PRKCI and miR-382-5p partially attenuated cell viability compared with alone circ-PRKCI overexpression in HPAEpiCs (Fig. S2A). Moreover, miR-382-5p mimic could rescue circ-PRKCI overexpression-inhibited Fe²⁺, ROS and MDA production in HPAEpiCs (Figs. S2B–S2E). In contrast, miR-382-5p mimic reversed circ-PRKCI overexpression-enhanced level of GSH in HPAEpiCs (Fig. S2F). In addition, ELISA data showed that the inhibition of IL-6, IL-1β and TNF-α by circ-PRKCI overexpression was partially reversed in HPAEpiCs after transfection of miR-382-5p mimic (Fig. S2G). Overall, our research demonstrated that circ-PRKCI relieves ferroptosis and inflammatory response in sepsis-induced ALI of HPAEpiCs by sponging miR-382-5p.