circ_0038467 promotes PM2.5-induced bronchial epithelial cell dysfunction

Abstract Purpose This study was to explore the toxicological mechanisms by which PM2.5 causes lung dysfunction. Methods The expression of circ_0038467 and miR-138-1-3p in PM2.5-induced human bronchial epithelial cell line BEAS-2B was detected by RT-qPCR. The effects of circ_0038467 and miR-138-1-3p on proliferation, apoptosis, and inflammatory cytokines (IL-6 and IL-8) in PM2.5-induced BEAS-2B were determined using cell counting kit-8, flow cytometry, western blot, and enzyme-linked immunosorbent assay, respectively. The levels of nuclear factor kappa B (NF-κB) pathway-related protein were also analyzed by western blot. The binding interaction between circ_0038467 and miR-138-1-3p was confirmed by dual-luciferase reporter assay and RNA immunoprecipitation assay and pull-down assay. Results circ_0038467 expression was increased by PM2.5 treatment in BEAS-2B cells in time- and dose-dependent methods, and knockdown of circ_0038467 reversed PM2.5-triggered BEAS-2B cell death and inflammatory response. miR-138-1-3p was decreased by PM2.5 treatment, and restoration of miR-138-1-3p attenuated PM2.5-induced BEAS-2B cell injury. In a mechanical study, we found circ_0038467 directly bound to miR-138-1-3p, and further rescue experiments exhibited miR-138-1-3p inhibition partially overturned the regulatory functions of circ_0038467 knockdown in PM2.5-induced BEAS-2B cells. Conclusion circ_0038467 provided a potential therapeutic strategy for future clinic intervention in air pollution-triggered lung dysfunction.


Introduction
Increasing evidence has shown that long-term air pollution can damage bronchial mucosa and induce bacterial invasion and inflammation reaction of airway and pulmonary, which lead to severe lung disorders in person; among them, the impact is particularly serious in children due to the incomplete development of the respiratory tract [1,2]. With the development of industry, air pollutant particle matters (PM) with an aerodynamic diameter less than 2.5 µm (PM2.5) have been revealed to be associated with the elevation of respiratory morbidity and mortality [3,4]. Compared with Western countries, the study into the hazard of PM2.5 in China has just started. PM2.5 is reported to mainly occur in the Beijing-Tianjin-Hebei Economic Zone, the Yangtze River Delta, the Pearl River Delta region, the three northeastern provinces, the Sichuan Basin, and other densely populated areas, and seriously affects public health both physically and emotionally [5]. PM2.5 is a mixture of solid and liquid particles, including black carbon, sulfate, nitrate, metals, polycyclic aromatic hydrocarbons, and automobile exhaust particles, with the characteristics of small particle size, large surface area, and toxin absorption ability [6]. It can be inhaled and deposited in the epithelium cells of the alveolar, thus aggravating airway inflammation, oxidative injury, aging, and immoderately death of cells [7][8][9]. Therefore, further investigations on the mechanisms underlying the toxicity of PM2.5 are necessary for developing effective therapies of lung dysfunction. Circular RNAs (circRNAs) are defined as a novel type of transcripts with ring structures from the ligation of exons, introns, or both that generally make them resist to RNase R decay [10,11]. circRNAs are abundantly expressed in the eukaryotic genome, exhibit species-, tissue-, cell-, and disease-specific expression patterns, and have been demonstrated to play key roles in cellular crucial biological processes [12][13][14]. Importantly, a growing number of studies have indicated that circRNAs are intensively associated with different respiratory diseases, such as lung cancer, pulmonary tuberculosis chronic obstructive pulmonary disease (COPD), and pulmonary hypertension [15,16]. circ_0038467 was a novel identified circRNA, produced from the back-splicing of ubiquinol-cytochrome-c reductase core protein 2 (UQCRC2) gene, and was found to be upregulated in lipopolysaccharide (LPS)-induced human bronchial epithelial (HBE) cells and reinforced LPSinduced cell inflammatory injury [17]. Thus, we suspected that circ_0038467 might also involve in PM2.5-induced HBE cell dysfunction.
Here, this work aimed to investigate the expression pattern and pathological role of circ_0038467 in PM2.5induced HBE cell line BEAS-2B and explored the potential regulatory network underlying circ_0038467 in PM2.5evoked BEAS-2B injury.
2 Materials and methods 2.1 PM preparation PM2.5 stock solution was prepared by dissolving standard reference PMs (SRM 1648a) (50 mg; NIST, Gaithersburg, MD, USA) in 500 µL phosphate buffer solution (PBS) and 500 µL dimethyl sulfoxide (DMSO) and subjected to ultrasound oscillation on ice for 30 min through ultrasonography. Then Toxinsensor™ Endotoxin Detection System (GenScript Biotech, Nanjing, China) was applied to test endotoxin levels.

RNA extraction and RNase R digestion
TRIzol reagent (Invitrogen) was used to isolate total RNA from BEAS-2B cells. Then 100 µg of RNA extracts were treated without or with RNase R (Qiagen, Tokyo, Japan) for 15 min at 37°C; thereafter, the resulting RNAs were purified by using RNeasy MinElute Cleanup Kit (Qiagen).

Lactate dehydrogenase release (LDH) assay
Following transfection, BEAS-2B cells (1 × 10 4 ) were exposed to 75 μg/mL PM2.5 for 48 h. Then the culture supernatants of BEAS-2B cells were collected and LDH release was detected using an LDH assay kit (Sigma-Aldrich) referring to the protocol's instructions.

Enzyme-linked immunosorbent assay (ELISA)
The concentrations of IL-6 and IL-8 from the culture supernatants of BEAS-2B cells after appropriate transfection and/or treatment were analyzed using commercial IL-6 and IL-8 ELISA kits (R&D Systems, Minneapolis, Minnesota, USA) referring to the instructions of protocol.

Subcellular fractionation
Cytoplasmic and Nuclear RNA Purification Kit (Thermo Fisher Scientific, Inc., Waltham, MA, USA) was employed to isolate and purify the RNA from nuclear and cytoplasm fractions. Next, the relative expression levels of circ_0038467, GAPDH, and U6 were determined by RT-qPCR assay.

RNA immunoprecipitation (RIP) assay
BEAS-2B cells were lysed by using RIP buffer, and then cell lysate was incubated with RIPA buffer containing magnetic beads conjugated with human anti-Ago2 antibody or normal mouse anti-IgG (Millipore, Billerica, MA, USA), followed by interaction with Proteinase K (Millipore). Finally, the immunoprecipitated RNA was extracted and circ_0038467 levels were analyzed using RT-qPCR assay.

Statistical analysis
Data from thrice-repeated experiments were presented as mean ± standard deviation (SD). Comparisons of parameters were analyzed using Student's t-test, Mann-Whitney U tests, and one-way analysis of variance (ANOVA) as appropriate.
The P < 0.05 was indicated as statistically significant.

PM2.5 accelerated circ_0038467 expression in BEAS-2B cells
To investigate the potential effects of circ_0038467 on PM2.5-induced injury of HBE cells, first of all, the different expression of circ_0038467 in HBE cells was detected. BEAS-2B cells were exposed to a range of concentrations of PM2.5 (0, 50, 75, and 100 µg/mL) for 48 h; the expression of circ_0038467 in HBE cells was found to be increased in a dose-dependent manner (Figure 1a). Moreover, circ_0038467 expression was also time-dependently upregulated in HBE cells by 75 µg/mL PM2.5 exposure at 0, 24, 48, and 72 h (Figure 1b). All these data suggested that aberrant expression of circ_0038467 might be associated with PM2.5-induced BEAS-2B cell dysfunction. Afterwards, RNase R assay was performed, and we found circ_0038467 was resistant to RNase R digestion, while the linear transcript was markedly digested by RNase R treatment (Figure 1c), indicating that circ_0038467 was indeed circular transcript.

circ_0038467 knockdown reduced PM2.5-induced cell death and inflammatory response in BEAS-2B cells
To get insights into the effect of circ_0038467 on PM2.5induced HBE cell injury, BEAS-2B cells were transfected with si-circ_0038467 or si-NC, then treated with 75 μg/mL PM2.5 for 48 h. The introduction of si-circ_0038467 obviously decreased PM2.5-induced upregulation of circ_0038467 expression (Figure 2a). From the CCK-8 assay, we found that PM2.5 exposure remarkably suppressed cell viability of BEAS-2B cells, while this condition was reversed by circ_0038467 knockdown (Figure 2b). LDH assay showed knockdown of circ_0038467 decreased PM2.5-induced LDH release in BEAS-2B cells (Figure 2c), revealing that downregulation of circ_0038467 suppressed PM2.5-induced damage to the membrane of BEAS-2B cells. Meanwhile, we verified that PM2.5 exposure increased the apoptosis rate of BEAS-2B cells by elevating Bax expression and decreasing Bcl-2 expression, while downregulation of circ_0038467 abated PM2.5-induced cell apoptosis (Figure 2d and e). Additionally, the results from ELISA and western blot analyses exhibited that PM2.5 exposure significantly increased IL-6 and IL-8 expression in BEAS-2B cells, which were attenuated by circ_0038467 silencing (Figure 2f-h). Taken together, downregulation of circ_0038467 blocked PM2.5-induced cell death and inflammatory response of BEAS-2B cells.

miR-138-1-3p was a target of circ_0038467
The subcellular localization of circ_0038467 was investigated, and results showed circ_0038467 was highly enriched in the cytoplasm fraction in BEAS-2B cells (Figure 3a). Besides, RIP assay suggested circ_0038467 could be enriched by anti-Ago2 antibody relative to IgG antibody (Figure 3b). Thus, we suspected that circ_0038467-mediated regulatory functions might operate through a sponge mechanism. Through the using of online databases CircInteractome, the potential underlying microRNAs (miRNAs) that could be interacted with circ_0038467 was searched, and miR-138-1-3p was identified to have putative binding sites of circ_0038467 (Figure 3c). Then dual-luciferase reporter assay was implemented in BEAS-2B cells, and we observed that the luciferase activity of wild-type reporter was significantly reduced by miR-138-1-3p overexpression, while there was no change in mutated reporter when miR-138-1-3p was upregulated (Figure 3d). Moreover, pull-down assay exhibited that circ_0038467 levels in BEAS-2B cells pulled down using Bio-miR-138-1-3p were higher than those in cells pulled down by Bio-miR-NC or Bio-miR-138-1-3p-MUT ( Figure 3e). All these results confirmed that circ_0038467 directly bound to miR-138-1-3p in BEAS-2B cells. Subsequently, the different expression of miR-138-1-3p in BEAS-2B cells was explored; RT-qPCR analysis suggested miR-138-1-3p expression was decreased by PM2.5 exposure (Figure 3f), but was increased by circ_0038467 knockdown (Figure 3g).

Discussion
Recently, inhalant PMs-induced air pollution is becoming continually intensified with the development of the heavy industries and lax control of the pollution sources, and it has been recognized that increased PM concentration in the air may directly cause growing morbidity of respiratory system [2,5]. However, the mechanisms by which PM2.5 results in lung dysfunction are still not systemically elucidated. In a series of recent studies, the role of circRNAs in PM2.5 toxic effects has begun to be identified.
In this study, we found PM2.5 treatment upregulated circ_0038467 expression in BEAS-2B cells. Then we transfected si-circ_0038467 into BEAS-2B cells before PM2.5 treatment, and results of functional experiments indicated circ_0038467 knockdown reversed PM2.5-induced cell viability inhibition, LDH release, as well as apoptosis promotion in BEAS-2B cells. Chronic inflammatory reaction triggered by PM2.5 deposition impairs lung function and increases the incidence of COPD and asthma [20]. This study provided experimental evidences showing that PM2.5 exposure triggered IL-8 and IL-6 expression upregulation, which was attenuated by circ_0038467 silencing. Taken together, we demonstrated that downregulation of circ_0038467 rescued lung function by blocking PM2.5-induced HBE cell death and inflammatory response.
Previous studies have documented that circRNAs can directly target microRNAs (miRNAs) by serving as miRNA sponges and subsequently abrogate the function of the corresponding miRNAs [21,22]. Given that circ_0038467 showed obvious stability and predominantly localized in the cytoplasm, we then investigated whether circ_0038467 performed regulatory effects by sponging miRNAs. Through using bioinformatics methods, we confirmed that miR-138-1-3p was a target of circ_0038467 in BEAS-2B cells. miRNAs are one of the small noncoding RNAs of about 22 nucleotides in length, which are implicated in posttranscriptional gene repression, and multiple studies have reported that miRNA alterations are associated with the development and progression of lung disease [23,24]. Several miRNAs, such as miR-486 and miR-331, have been demonstrated to circ_0038467 on bronchial epithelial cell injury  861 inhibit PM2.5-induced cell apoptosis, oxidative stress, and inflammation in human airway epithelial cells [25,26]. However, the roles of miR-138-1-3p in PM2.5 causing lung dysfunction are still unknown.
The NF-κB pathway plays a key role in regulating important cellular behaviors, particularly, cellular growth, apoptosis, and inflammatory responses, deregulation of which is associated with cancer, inflammatory, autoimmune diseases, and so on [27][28][29]. Interestingly, recent evidence uncovered that PM2.5 exposure could activate the NF-κB pathway to induce inflammation and uncontrolled proliferation in human HBE cells, thus inducing lung dysfunction [26,30,31]. In the present study, we also found that PM2.5 exposure could activate NF-κB pathway via elevating p-IκB-α and p-p65 levels, while this condition was impaired by circ_0038467 knockdown. Importantly, we also demonstrated that miR-138-1-3p inhibition overturned the inhibitory effects of si-circ_0038467 on p-IκB-α and p-p65 expression under PM2.5 exposure. Overall, PM2.5 mediated the activation of NF-κB pathway via circ_0038467/ miR-138-1-3p axis in HBE cells.

Conclusion
In summary, this study uncovered that knockdown of circ_0038467 mitigated PM2.5-induced HBE cells death and inflammation via regulating miR-138-1-3p/NF-κB pathway, providing new insights into the molecular toxicological mechanisms underlying the PM2.5 and potential therapeutic strategies for future clinic intervention in PM2.5induced lung disorders.
Funding information: No funding was received.
Authors contribution: All authors made substantial contribution to conception and design, acquisition of the data, or analysis and interpretation of the data; take part in drafting the article or revising it critically for important intellectual content; gave final approval of the revision to be published; and agree to be accountable for all aspect of the work.