Circ_0035292 knockdown alleviates lipopolysaccharide (LPS)‐induced WI‐38 cell apoptosis and inflammatory injury

Abstract Background Circular RNAs have emerged as important regulators in the pathogenesis of human diseases, including infantile pneumonia (IP). In this study, we aimed to explore the effects of circ_0035292 on lipopolysaccharide (LPS)‐treated Wistsar Institute (WI)‐38 cells. Methods Quantitative real‐time polymerase chain reaction and western blot were executed to detect the levels of circ_0035292, microRNA‐370‐3p (miR‐370‐3p) and transducin β‐like 1X related protein 1 (TBL1XR1). Cell counting kit‐8, 5‐ethynyl‐2′‐deoxyuridine, and flow cytometry assessed cell proliferation and apoptosis. Concentrations of inflammatory factors were examined with enzyme linked immunosorbent assay kits. Dual‐luciferase reporter assay and RNA immunoprecipitation were adopted to analyze binding between miR‐370‐3p and circ_0035292 or TBL1XR1. Results Circ_0035292 level was increased in IP patients and LPS‐triggered WI‐38 cells. Circ_0035292 knockdown rescued LPS‐mediated WI‐38 cell proliferation suppression and WI‐38 cell apoptosis and inflammation promotion. Circ_0035292 interacted with miR‐370‐3p and miR‐370‐3p directly targeted TBL1XR1. Moreover, miR‐370‐3p overexpression alleviated LPS‐induced WI‐38 cell apoptosis and inflammatory injury, which was abrogated via TBL1XR1 upregulation. Circ_0035292 absence inhibited the NF‐κB pathway. Conclusion Knockdown of circ_0035292 rescued LPS‐triggered WI‐38 cell injury via miR‐370‐3p/TBL1XR1 axis and NF‐κB pathway.


| INTRODUCTION
Infantile pneumonia (IP) is one of the most common infectious diseases in infants and young children. 1,2 IP is mainly caused by bacterial or viral infection, posing a huge challenge to infants and young children's health. 1 The current approach to IP is antiviral, antibacterial, and symptomatic supportive therapy. 3 However, the continuous usage on antibiotics has led to an increasing resistance of pathogenic bacteria, making the disease difficult to cure. 4 Meanwhile, the severe inflammatory reaction can damage the respiratory system of infants and young children. 5 Therefore, further knowledge of the underlying mechanism of IP is essential to IP therapy.
During the past decades, as a large class of primarily noncoding RNA, circular RNAs (circRNAs) undergo backsplicing events to form covalently closed-loop structures. 6 In mechanism, some of them can act as ceRNAs to sponge for miRNAs, thus regulating gene content. 7 Several laboratory works discovered that dysregulated circRNAs might be implicated in the pathogenesis of pneumonia. 8,9 For example, circ-UQCRC2 absence alleviated lipopolysaccharide (LPS)-triggered 16HBE cell inflammation in IP via MYD88 expression. 10 Circ_VMA21 receded LPS-initiated Wistsar Institute (WI)-38 cell injury via enhancing KLF4 through sponging miR-409-3p. 11 Moreover, circ_0035292 (circTMOD3) interference was able to relieve LPS-caused cell injury via miR-146b-3p/CXCR1 pathway. 12 Even so, circ_0035292-mediated potential regulatory mechanisms in IP are largely elusive.

| Sample acquisition
After the Ethics Committee of Wuhan Asia General Hospital authorized the study and the participants provided written informed consents, the blood samples were provided by 23 IP subjects and 19 volunteers at this hospital. Serum samples were stored at −80°C after centrifugation at 1000g.

| Quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNA was prepared according to TRIzol (Beyotime). After that, RNAs was used for cDNAs generation experiments based on PrimeScript™ RT reagent Kit (Takara) or miRNA 1st Strand cDNA Synthesis Kit (Vazyme). qRT-PCR was executed using AceQ Universal SYBR qPCR Master Mix (Vazyme) with primer sequences ( Table 1). The expression was estimated with 2 C -ΔΔ t strategy and normalization to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) or U6. Additionally, to analyze the stability of circ_0035292, RNase R exposure at 37°C was applied for RNAs. Circ_0035292 and GAPDH content was determined.

| Flow cytometry
Apoptosis of WI-38 cells was verified according to the steps of the Annexin V-fluorescein isothiocyanate (V-FITC)/PI kit (Beyotime). The cells were collected, washed with PBS and repeated once and removed, the cells were resuspended by adding the kit's buffer again, followed by the addition of 5 μL Annexin V-FITC and 5 μL PI, mixed and placed at 37°C for 15 min, and cell apoptosis was verified according to flow cytometry.

| Western blot
Referring to RIPA buffer (Beyotime), proteins in serums and cells were prepared. After separation on 10% SDS-PAGE gel, proteins were blotted onto PVDF membranes, which were incubated with primary antibodies and interacted with secondary antibody. The end of the experiment, ECL reagent (Beyotime) was utilized for protein bands. Abcam provided secondary antibody (ab6728) and primary antibodies BCL2-Associated X (Bax; ab32503), B-cell lymphoma-

| Dual-luciferase reporter
Fragments of circ_0035292 and TBL1XR1 containing binding sequences of miR-370-3p were introduced into pmirGLO (Promega) to construct the WT-circ_0035292/ TBL1XR1. MUT-circ_0035292 and MUT-TBL1XR1 were constructed via mutating the binding sequences. miR-370-3p mimic/miR-NC and generated vectors were cotransfected into WI-38 cells. After 48 h of transfection, the culture medium was changed. Cells were collected and lysed after 24 h. After centrifugation at 3500 r/min for 5 min, the supernatant was removed and sample was monitored based on Dual Luciferase Activity Assay Kit (Promega).

| RIP
After RNA Immunoprecipitation (RIP) buffer lysis (EMD Millipore), samples were mixed with anti-IgG or anti-Ago2-conjugated magnetic beads. The immunoprecipitated RNAs were determined by qRT-PCR.

| Statistical analysis
Software GraphPad Prism 7 analyzed data, which were presented as mean ± SD. Difference analysis was conducted via Student's t-test or one-way analysis of variance (ANOVA). Linear relationships were analyzed by Spearman's correlation coefficient analysis. It was considered significant if p < .05.

| LPS treatment hindered WI-38 cell proliferation and promoted apoptosis and inflammation
First of all, WI-38 cells were treated with different doses of LPS (0, 5, 10, and 15 μg/mL) for 12 h. As a result, LPS diminished WI-38 cell viability in a dose-dependent manner ( Figure 1A). Simultaneously, applying LPS dampened WI-38 cell proliferation ( Figure 1B). On the contrary, WI-38 cell apoptosis was facilitated via LPS exposure ( Figure 1C). Moreover, LPS exposure increased Bax level and reduced Bcl-2 in WI-38 cells in a dosedependent manner ( Figure 1D,E). As displayed in Figure 1F−H, LPS treatment increased IL-6, IL-1β and TNF-α concentrations. To sum up, LPS treatment caused fibroblast cell injury.

| Circ_0035292 knockdown promoted LPS-stimulated WI-38 damage
As shown in Figure 2A, circ_0035292 level was upregulated in serums of IP patients. Consistent with other typical circRNAs, circ_0035292 was resistant to RNase R digestion ( Figure 2B). Circ_0035292 level was elevated in LPS-treated WI-38 cells in a dose-dependent WI-38 cells, while circ_0035292 knockdown restored the effect ( Figure 2H and Supporting Information: Figure S1). In addition, LPS-induced IL-6, IL-1β and TNF-α elevation was ameliorated by circ_0035292 knockdown in WI-38 cells ( Figure 2I−K). In addition, circ_0035292 level was positively correlated with these inflammatory factors in IP subjects (Supporting Information: Figure S2). Taken together, circ_0035292 inhibited LPS-caused WI-38 cell injury.

| Circ_0035292 targeted miR-370-3p
Furthermore, potential target miRNAs of circ_0035292 were analyzed by circinteractome. As presented in Figure 3A, there are continuous binding sites in the nucleotide sequences of miR-370-3p and circ_0035292.

| DISCUSSION
The high incidence of pneumonia represents a significant challenge to public health worldwide. 18 Inducing an inflammatory response, LPS has been extensively utilized to build IP model in vitro. 19,20 CircRNAs are vital regulators in pneumonia progression. 8 Up to date, only a few circRNAs in pneumonia have been discovered. In the present research, the functions of circ_0035292 in IP were investigated using LPS-evoked WI-38 cells. Numerous circRNAs play vital regulatory roles in pneumonia process. For instance, circ_0038467 interference restored LPS-induced apoptosis and inflammatory damage of MRC-5 cells through regulating miR-195-5p/ TLR4 pathway and inactivating NF-κB pathway. 21 Circ_0038427 deficiency undermined LPS-aroused inflammatory damage via decoying miR-338-3p. 22 LPStriggered WI-38 cell inflammation and apoptosis might be at least partially alleviated via circ_0035292 absence. 12 Herein, circ_0035292 was abnormally increased in IP subjects. Its deficiency might overturn LPS-elicited WI-38 cell proliferation repression and apoptosis and inflammatory promotion.
Interestingly, there have several literature exhibiting that NF-κB pathway is associated with inflammatory response in pneumonia. 21,29 Accordingly, influence of circ_0035292 on NF-κB pathway was explored. Our results exhibited that circ_0035292 knockdown inhibited LPS-induced activation of NF-κB pathway through the interaction with miR-370-3p/TBL1XR1. However, the present project was limited in vitro studies, and we will use a murine model to explore the novel mechanism in IP in the further.
In conclusion, circ_0035292 aggravated LPS-triggered WI-38 cell damage through miR-370-3p/TBL1XR1/ NF-κB pathway. Our study provided a novel pathological mechanism in IP and might be useful for IP therapy.

AUTHOR CONTRIBUTIONS
Ying Guo, Zhouzhen Li, and Chen Cheng designed the concepts. Ying Guo and Zhouzhen Li performed the experiments. Ying Guo and Zhouzhen Li acquired and analyzed the data. Ying Guo and Zhouzhen Li prepared and edited the manuscript. Chen Cheng supervised the study and reviewed the manuscript. All authors approved the final edition.