CircVAMP3 promotes acute lung injury progression by regulating the miR-580-3p/STING/IFN-β axis in macrophages

Cell culture and treatment

THP-1 cells, a human monocytic cell line, were obtained from Fuheng (Guangzhou, China) and cultured in the Roswell Park Memorial Institute-1640 (Cat. No. 11875093 Gibco, Waltham, MA, USA) medium supplemented with 10% fetal bovine serum (Cat. No. F0850 Merck, Rahway, NJ, USA) under standard culture conditions (37 °C, 5% CO₂, and 95% humidity). To induce differentiation into macrophages, THP-1 cells were seeded in six-well plates and treated with phorbol 12-myristate 13-acetate (PMA, Cat. No. S7791 Selleck, Houston, TX, USA) at a final concentration of 100 nM for 48 h. Following differentiation, cells were washed with phosphate-buffered saline (PBS, Cat. No. G4202 Servicebio, Hubei, China) and allowed to rest in complete culture medium for 6 h prior to further treatment.

To establish an in vitro ALI model, THP-1-derived macrophages were stimulated with Escherichia coli-derived lipopolysaccharide (LPS, Cat. No. L2880 Sigma, St. Louis, MO, USA) at a final concentration of 1,000 ng/mL for 6 h, while the control group received an equal volume of PBS. Each experimental condition was conducted in triplicate to ensure reproducibility.

CircRNA identification and RNase R treatment

Total RNA of cells was extracted from cells using TRIzol reagent (Cat. No. 15596018CN Thermo Fisher, Waltham, MA, USA) following the manufacturer’s protocol, ensuring that samples were neither frozen nor chemically fixed prior to extraction. The quality and concentration of the extracted RNA was assessed using a NanoDrop spectrophotometer (Thermo Fisher, Waltham, MA, USA), measuring absorbance at 260 nm to determine RNA concentration. The purity of RNA was evaluated by calculating the ratio of absorbance at 260 nm and 280 nm (A260/A280), with a ratio between 1.8 and 2.0 considered indicative of high-quality RNA.

For reverse transcription, one µg of total RNA was used to synthesize complementary DNA (cDNA) using a commercially available kit (Cat. No. RR036A Takara Bio, Shiga, Japan) on a ProFlex PCR System (Thermo Fisher, Waltham, MA, USA). We amplified cDNA using TB Green Premix Ex Taq™ II (Cat. No. RR42WR Takara Bio, Shiga, Japan) with specific primers.

For RNase R digestion, 1∼10 µg of total RNA was incubated in a 20 µL reaction mixture containing 4 U RNase R per µg of RNA (NC1163864, Lucigen, Middleton, WI, USA) and two µL of enzyme buffer. The reaction was carried out at 37 °C for 15 min, followed by RNA purification using phenol-chloroform extraction to remove enzyme and reaction byproducts. The products were separated on a 2% agarose gel containing Nucleic Acid Red as a nucleic acid stain and visualized under UV illumination at 365 nm which were subjected to Sanger sequencing to identify the back splicing site.

Quantitative real-time PCR

Quantitative real-time PCR (qRT-PCR) was performed to quantify the expression levels of circRNAs and mRNAs encoding proteins or cytokines using the QuantStudio 7 Flex Real-Time PCR System (Thermo Fisher, Waltham, MA, USA) using the SYBR Green (Roche Group, Basel, Switzerland) according to the manufacturer’s protocol. Each reaction was prepared in a final volume of 20 µL, containing two µL of cDNA template, 10 µL of SYBR Green (Cat. No. 11200ES Yeasen Bio, Shanghai, China), and 0.4 µM of each forward and reverse primer. The cycling conditions included an initial denaturation at 95 ° C for 30 s, followed by 40 cycles of denaturation at 95 °C for 10 s and annealing/extension at 60 °C for 30 s. A melt curve analysis was performed to confirm the specificity of amplification. The primers for the candidate genes or circRNAs were listed in Table 1. GAPDH gene was selected as the internal reference gene. 2^−ΔΔCT method was used to statistically analyze the RT-qPCR data. Each experiment included a minimum of three biological replicates per sample to ensure reproducibility.

RNA oligo transfection

Cells were transfected with RNA oligo (synthesized by Tsingke, Shanghai, China) using Lipofectamine 2000 (Cat. No. 11668019 Thermo Fisher, Waltham, MA, USA), according to the manufacturer’s instructions. Briefly, RNA oligos were mixed with Lipofectamine 2000 reagent in Opti-MEM (Cat. No. 31985070 Thermo Fisher, Waltham, MA, USA) and incubated for 20 min at room temperature to allow complex formation. The transfection mixture was then added to cells in a 6-well plate, and the medium was replaced with fresh complete medium 6 h post-transfection. Cells were harvested after another 48 h, unless differently specified. A detailed list of the RNA oligo used in this study is provided in Table 2.

Lentivirus transfection

Lenti-circVAMP3 was synthesized and cloned by Genechem Co., Ltd. (Shanghai, China), and Lenti-NC was used as the negative control. For transfection, 2 × 10⁵ cells per well were seeded into 24-well plates 24 h prior to transfection, ensuring that the cells reached was reached 70∼80% confluence at the time of transfection. Transfection was performed using HiTransG A (Cat. No. REVG004 Genechem, Shanghai, China) according to the manufacturer’s instructions. After transfection, cells were cultured for 48 h before being subjected to puromycin (Cat. No. 60209ES10, Yeasen, Shanghai, China) selection at a concentration optimized for the cell line. Cells were cultured under selection until no uninfected cells remained, and only the successfully transduced cells survived. The transduced cells were then harvested for subsequent experiments.

Statistical analysis

All datasets were analyzed using GraphPad Prism (Version 10.3.0, GraphPad Software, LLC., Boston, MA, USA). Quantitative data, derived from a minimum of three independent biological replicates, were reported as mean ± standard deviation (SD) following validation of parametric assumptions. Briefly, normality was confirmed via Shapiro–Wilk testing, and variance homogeneity was assessed using Brown-Forsythe tests. Intergroup comparisons were conducted with two-tailed unpaired Student’s t-tests, while multigroup analyses utilized one-way ANOVA preceded by Bartlett’s test for variance homogeneity. Statistical significance was set at *P < 0.05.

circVAMP3 expression was significantly elevated in macrophage ALI model

We constructed an ALI model through LPS stimulation PMA-differentiated THP-1 macrophages. Subsequent qRT-PCR validation at the cellular level revealed distinct circRNA expression profiles, with concomitant elevation of IL-1β and IFN-β levels confirming successful model induction (Fig. 1A). Notably, circVAMP3 (circRNA.1_7777160_7778169, hsa_circ_0006354) emerged as the exclusively upregulated circRNA in this macrophage-based ALI system (Fig. 1B). To further exclude the effect of VAMP3 linear RNA, we examined the linear RNA using sequence primers not present in circVAMP3 (in exon 5) and found that mRNA did not change significantly in the same model (Fig. 1C). Covalently closed circular structure was confirmed by qRT-PCR and gel electrophoresis on RNA either untreated (RNase R (-)) or treated (RNase R (+)) with the RNase R exonuclease. Linear RNA demonstrated significant alterations, whereas circRNA maintained structural stability (Figs. 1D, 1E). Then circVAMP3 primer amplification product was purified and submitted to Sanger sequencing, and the results verified that back-splicing junction was elevated in amplified product (Fig. 1F), confirming the changed VAMP3 expression was a circular RNA formed by reverse splicing of exons 3 and 4 rather than linear mRNA (Fig. 1G). Therefore, we speculated that circVAMP3 might be a potential regulator of pathophysiological processes in ALI/ARDS.

CircVAMP3 regulates expression of IFN-β in macrophage ALI model

To elucidate the role of circVAMP3 in macrophage inflammatory responses during ALI pathogenesis, we knocked circVAMP3 down through siRNA and overexpressed through lentivirus. After successfully knockdown of circVAMP3, we found that IL-1β, a classical proinflammatory cytokine, remained unaffected, while IFN-β, a type 1 interferon primarily activated in macrophages, was significantly down-regulated under si-circVAMP3 (Fig. 2A). This specificity was further corroborated where lentiviral-driven circVAMP3 overexpression significantly potentiated IFN-β expression without altering IL-1β levels (Fig. 2B), suggesting that circVAMP3 may regulate the expression of IFN-β in macrophages in a certain specific way.

CircVAMP3 regulates the expression of IFN-β through STING in macrophage ALI model

To delineate how CircVAMP3 regulates the expression of IFN-β, we studied the expression levels of major pathway proteins that regulate IFN expression on mRNA levels including cGAS-STING and MDA5/RIG-1 in macrophage ALI model. Our experimental results showed that in macrophage ALI model, the mRNA expression of cGAS and STING was significantly increased, and the expression of MDA5 was also increased, while there was no significant change in RIG-1 expression (Fig. 3A).

To further study which protein CircVAMP3 targets, we examined the three proteins in cells which down-regulated and up-regulated CircVAMP3. We found that when transfected with si-CircVAMP3 to down-regulated the expression level, only STING showed statistically significant downregulation synchronously, while cGAS and MDA5 did not change significantly (Fig. 3B). When we successfully transfected lenti-CircVAMP3 to up-regulate CircVAMP3, only STING expression level increased with the up-regulation of CircVAMP3, and there was no statistical difference in cGAS and MDA5 expression level (Fig. 3C), which was consistent with the results of siRNA knockdown, suggesting CircVAMP3 might selectively modulates STING transcription

CircVAMP3 regulates STING expression through miR-580-3p

In our previous experimental section, we had found that CircVAMP3 might regulates IFN-β expression by influencing STING levels. Considering circRNA plays a role in the regulation of gene expression mainly through sponge binding specific miRNA, we speculated that there would be a miRNA regulated by CircVAMP3 and regulating the mRNA of STING. We used the CircInteractome database (https://circinteractome.nia.nih.gov/) to predict target miRNA of CircVAMP3 and TargetScanHuman (https://www.targetscan.org/vert_72/) to predict miRNA which can regulate mRNA of STING. We found a total of seven miRNAs that CircVAMP3 could target and regulate, and 301 miRNAs that could target and regulate STING. Among the miRNAs obtained by databases, miR-580-3p is the only miRNA that is regulated by CircVAMP3, and regulate STING mRNA (Fig. 4).

To verify the role of miR-580-3p, we studied the expression levels in macrophage ALI models and CircVAMP3 intervened models. The results showed that miR-580-3p was significantly down-regulated in macrophage ALI model compared with the control group and in the CircVAMP3 intervention model, down-regulation of CircVAMP3 expression mediated by si-CircVAMP3 led to the increase of miR-580-3p; up-regulation of CircVAMP3 induced by lenti-CircVAMP3 inhibited the expression level of miR-580-3p (Fig. 5A). The consistent results suggest that CircVAMP3 can inhibit the expression level of miR-580-3p, and LPS can regulate miR-580-3p by regulating CircVAMP3.

After confirming the correlation between CircVAMP3 and miR-580-3p, we further studied the correlation between miR-580-3p and STING. We regulated the effect of miR-580-3p through miRNA mimic and inhibitor to observe whether it had an effect on the expression of STING/IFN-β. Results showed that when miRNA concentration was increased by miR-580-3p mimic, STING expression was significantly down-regulated in mRNA levels, accompanied by simultaneous down-regulated levels of IFN-β. The expression level of cGAS was not affected (Fig. 5B). However, after the biological effects of miR-580-3p were inhibited by inhibitor, the expression level of STING was increased, accompanied by the simultaneous increase in the expression level of IFN-β (Fig. 5C), suggesting that miR-580-3p could bind STING mRNA. So far, our experiments have proved that CircVAMP3 can inhibit the miRNA effect of miR-580-3p in macrophage ALI model, thereby up-regulating the expression level of STING and promoting the expression of IFN-β.