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Introduction

Pneumonia is a common inflammation-related disease with high morbidity and mortality rates among infectious diseases and is an important health problem worldwide. Persistent inflammatory infection with pneumonia can induce lung tissue damage (1,2). The drugs commonly administered in the clinical treatment of pneumonia include antibiotics and adrenocorticoid hormones (3). However, after treatment with these drugs, the body will develop resistance and significant side effects including gastrointestinal infections and metabolic disturbance (4,5). Therefore, it has become an urgent problem for researchers to improve the treatment efficacy of pneumonia and to understand the underlying mechanism of the disease.

Under pathological conditions, the environment in the endoplasmic reticulum (ER) changes and misfolded and unfolded proteins accumulate in the ER cavity until the ER homeostasis is disrupted. Following this, a series of stress reactions in the ER are triggered to process these misfolded or unfolded proteins and maintain the normal function of cells. This process is called endoplasmic reticulum stress (ERS) (6). Hypoxia, inflammatory response, oxidative stress and other interfering factors can lead to the increase of protein misfolding rate and protein load in the ER, inducing ERS (7). A previous study showed that alleviating inflammation and ERS in pediatric pneumonia can significantly inhibit the development of pediatric pneumonia (8). Therefore, it is beneficial to improve the symptoms of pneumonia by alleviating ERS and inflammation. Previous research has shown that Stromal interaction molecule 1 (STIM1)-Orai1 interaction exacerbates LPS-induced inflammation and ERS in bovine hepatocytes through store-operated calcium entry (9). STIM1 is a recently discovered vasoactive protein that can participate in the regulation of inflammatory processes (10-13). A previous study has shown that STIM1 is closely related to activation of the inflammatory response and platelet aggregation after stent implantation in percutaneous coronary intervention (14). In addition, overexpression of SOX9 can alleviate the inflammatory damage of bronchial epithelial cells induced by cigarette smoke extract by inhibiting STIM1 (15). Furthermore, silencing STIM1 expression inactivates NLR pyrin domain containing 3 by promoting the expression of microRNA-223, thereby reducing the inflammatory damage of lung epithelial cells induced by influenza A virus (16). STIM1 has also been shown to reduce lipopolysaccharide (LPS)-induced inflammation by inhibiting NF-κB signaling in bovine mammary epithelial cells (17). However, there are few studies investigating the regulatory role of STIM1 on ERS and inflammation in pneumonia.

The present study predicted the potential interaction of the RNA-binding protein insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2) with STIM1 via RBPmap website. IGF2BP2 belongs to a highly conserved family of RNA-binding proteins whose function is to regulate mRNA localization, stability and translation and fine-tune the physiological function of the encoded protein (18). IGF2BP2 knockdown inhibited LPS-induced inflammation of lung epithelial cells by targeting caspase 4, thus inhibiting the non-standard scorching pathway (19). So it was hypothesized that IGF2BP2 can regulate STIM1 and thus play a role in LPS-induced pneumonia.

In the present study, the role of STIM1 in an LPS-induced pneumonia cell model and its regulatory mechanism were investigated, to provide a useful theoretical basis for the future clinical treatment of pneumonia.

Materials and methods

Database

The RNA-binding protein IGF2BP2 has a potential interaction with STIM1, which was investigated using the web-tool RBPmap (http://rbpmap.technion.ac.il/) that enables prediction of RBP binding on genome sequences from a huge list of experimentally validated motifs of RBPmap database (Table SI) (20).

Cell culture

The human lung adenocarcinoma epithelial cells A549 purchased from the BeNa Culture Collection (cat. no. BNCC337696) and was cultured in Dulbecco's Modified Eagle's Medium (DMEM; Thermo Fisher Scientific, Inc.) containing 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) in a 37˚C incubator with 5% carbon dioxide. For the injury model, the cells were placed in 6-well plates (3x105 cells/well) and incubated for 12 h, before incubation with LPS (10 µg/ml) for a further 24 h (21). LPS induces the human pulmonary epithelial cell line A549 to create a pneumonia model (22,23).

Reverse transcription quantitative PCR (RT-qPCR)

The A549 cells in a 6-well plate at a density of 2x105 cells/well were lysed using TRIzol® reagent (Thermo Fisher Scientific, Inc.) and total RNA was purified using a RNeasy Plus Mini Kit (Qiagen, Inc.). Then, cDNA was generated using 1 µg total RNA and a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. The cDNA was then amplified by IQTM SYBR Green Master Mix (Bio-Rad Laboratories, Inc.), which was then quantified using a SYBR-Green detection system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The following thermocycling conditions were used for the qPCR: Initial denaturation at 95˚C for 10 min; 50 cycles of 95˚C for 15 sec and 60˚C for 60 sec. Each reaction was performed three times. Finally, the 2-ΔΔCq quantification method was used to calculate the expression levels of target mRNAs (24). The GAPDH mRNA level was used as the normalized standard. The PCR primers were as follows: STIM1 forward: 5'-GCCTAGGAGGCCCAGGAT-3', reverse: 5'-ACAGCCAAAGGTCAAGTGCT-3'; IGF2BP2 forward: 5'-GGAACAAGTCAACACAGACACA-3', reverse: 5'-CGCAGCGGGAAATCAATCTG-3'; GAPDH forward: 5'-AATGGGCAGCCGTTAGGAAA-3', reverse: 5'-GCGCCCAATACGACCAAATC-3'.

Western blotting

The A549 cells were lysed in RIPA buffer and protein contents were determined by the BCA method. In total, 20 µg of proteins was separated by 12% SDS-PAGE and then transferred to PVDF membranes. The membranes blocked with 5% BSA (Sigma-Aldrich; Merck KGaA) for 1 h at room temperature were incubated with primary antibodies STIM1 (1:1,000; cat. no. ab108994; Abcam), Bcl-2 (1:1,000; cat. no. ab182858; Abcam), Bax (1:1,000; cat. no. ab32503; Abcam), glucose-regulated protein 78 (GRP78; 1:1,000; cat. no. ab21685; Abcam), activating transcription factor 6 (ATF6; 1:1,000; cat. no. ab227830; Abcam), C/EBP homologous protein (CHOP) (1:1,000; cat. no. 5554; CST), caspase 12 (1:1,000; cat. no. ab62484; Abcam), PKR-like ER kinase (PERK; 1:1,000; cat. no. ab229912; Abcam), insulin-like growth factor 2 mRNA binding protein 3 (IGF2BP3; 1:1,000; cat. no. ab177477; Abcam), phosphorylated (p-)PERK (1:1,000; cat. no. 3179; CST), GAPDH (1:1,000; cat. no. ab9485; Abcam) overnight at 4˚C. Then, the appropriate horseradish peroxidase-conjugated secondary antibody (1:5,000; cat. no. ab150077; Abcam) was incubated with the membranes for 1 h at 37˚C. GAPDH was used as the internal control. Protein bands were then detected by enhanced chemiluminescence (MilliporeSigma). The western blot images were analyzed using Image J software (V1.8.0, National Institutes of Health).

Cell transfection

Insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2) overexpression lentivirus (Oe-IGF2BP2) and the corresponding negative control (Oe-NC; pcDNA3.1) and small interfering (si)RNAs against STIM1 (si-STIM1#1 and si-STIM1#2) and their corresponding scrambled sequence negative control (si-NC) were obtained from Shanghai GenePharma Co., Ltd. Transfection was conducted at 37˚C for 48 h using Lipofectamine® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. The transfection efficiency was detected by RT-qPCR or western blotting as aforementioned 48 h after transfection. The sequences were as follows: si-STIM1#1 sense, 5'-UCAAUUCGGCAAAACUCUGCU-3' and antisense, 5'-CAGAGUUUUGCCGAAUUGACA-3'; si-STIM1#2 sense, 5'-UCAGUUUGUGGAUGUUACGGA-3' and antisense, 5'-CGUAACAUCCACAAACUGAUG-3'; si-IGF2BP2#1 sense, 5'-AGUAGUUCUCAAACUGAUGCC-3' and antisense, 5'-CAUCAGUUUGAGAACUACUCC-3'; si-IGF2BP2#2 sense, 5'-UCUUGAAGGAGUAGUUCUCAA-3' and antisense, 5'-GAGAACUACUCCUUCAAGAUU-3'; and si-NC sense, 5'-UUCUCCGAACGUGUCACGUTT-3' and antisense, 5'-ACGUGACACGUUCGGAGAATT-3'.

Cell Counting Kit (CCK)-8 assay

A549 cells were seeded into 96-well plates at a density of 5x103 cells/well and were treated accordingly before incubation with 10 µl CCK-8 working solution (MedChemExpress) for 4 h. Absorbance was measured at 450 nm using a microplate reader.

Flow cytometry

A549 cells were seeded into 96-well plates at a density of 2x106 cells/well. After the corresponding incubation, cells were stained with FITC-conjugated annexin V and propidium iodide using an annexin V-FITC apoptosis kit (Beyotime Institute of Biotechnology) for 10 min in the dark at room temperature. Apoptotic cells were quantified by loading the cell mixture onto a CytoFLEX flow cytometry system (Beckman Coulter, Inc.) and FlowJo software (Version 10; FlowJo LLC) was used to analyze the data. Apoptosis rate was calculated as the sum of the early apoptosis rate (the lower right quadrant) and the late apoptosis rate (the upper right quadrant).

Enzyme-linked immunosorbent assay (ELISA)

To detect the levels of IL-6 (cat. no. ab178013; Abcam), IL-1β (cat. no. ab214025; Abcam) and TNF-α (cat. no. ab181421; Abcam) in the cell supernatants, related ELISA assay kits were used according to the manufacturer's recommendations.

Immunofluorescence (IF)

After treatment, A549 cells were fixed in 4% paraformaldehyde (300 µl/well; Sigma-Aldrich; Merck KGaA), incubated in 0.3% Triton X-100 (500 µl/well; Sigma-Aldrich; Merck KGaA) for 15 min and then blocked with 5% goat serum (Sigma-Aldrich; Merck KGaA). The slides were then stained with CHOP primary antibody (1:300; cat. no. 5554; CST) overnight at 4˚C and the fluorescent secondary antibody (1:500; cat. no. ab150077; Abcam) at 37˚C for 30 min, before being stained with DAPI (MilliporeSigma) for 10 min at room temperature in mounting medium. The cells were observed using an LSM 710 confocal laser microscope system (Carl Zeiss AG).

RNA-binding protein immunoprecipitation (RIP) assay

RIP assays were performed according to the EZ-Magna RIP RNA-binding Protein Immunoprecipitation Kit (MilliporeSigma). The cells were collected and added with 10 ml PBS and 0.01% formaldehyde to crosslink for 15 min. Then 1.4 ml 2 mol/l glycine was added and mixed for 5 min before being centrifuged for 5 min at 1,000 x g at room temperature. After discarding the supernatant, the cells were lysed with RIPA lysis buffer with 20 µl of protein A resin addition and incubated for 1 h at 4˚C. The supernatant was discarded and 50 µl PBS was added for suspension. Total RNA was extracted from suspension with TRIzol® reagent and reverse synthesis of cDNA. Finally, STIM1 mRNA abundance was detected by RT-qPCR as aforementioned.

RNA stability analysis

After treatment, A549 cells were exposed to 2 µg/ml actinomycin D (Cayman Chemical Company) for 0, 4, 8, 12 or 24 h to block transcription. DMSO (MilliporeSigma) was used as the control reagent. Cell samples were collected at the indicated time points and RNA was extracted. Finally, STIM1 mRNA expression levels were detected by RT-qPCR as aforementioned (25).

Statistical analysis

The data are presented as the mean ± SD and were analyzed using GraphPad Prism 5 (Dotmatics). The comparisons were assessed using one-way ANOVA followed by Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference. Each experiment was repeated at least three times.

Results

STIM1 is upregulated in LPS-induced A549 cells and STIM1 knockdown inhibits LPS-induced A549 cell apoptosis

After A549 cells were induced with LPS, the expression of STIM1 was detected by RT-qPCR and western blotting. The results demonstrated that the expression of STIM1 was significantly increased after LPS induction, compared with the untreated control cells (Fig. 1A and B). Following this, STIM1 interference plasmids were constructed, A549 cells were transfected with these plasmids and the transfection efficacy was detected by RT-qPCR and western blotting (Fig. 1C and D). si-STIM1#1 was selected for follow-up experiments due to its more prominent interference efficacy. Cells were then divided into the control, LPS, LPS + si-NC and LPS + si-STIM1 groups. Subsequently, cell viability was measured by CCK-8 and the results demonstrated that cell viability was significantly decreased in the LPS group compared with the control group. Furthermore, compared with the LPS + si-NC group, cell viability was increased in the LPS + si-STIM1 group (Fig. 1E). Flow cytometry demonstrated that LPS treatment induced cell apoptosis, which was significantly reduced after inhibition of STIM1 expression (Fig. 1F). Western blotting analysis of apoptosis-related proteins demonstrated that Bcl-2 expression was decreased and Bax expression was increased in the LPS group, compared with the control group. Furthermore, compared with the LPS + si-NC group, Bcl-2 expression was increased and Bax expression was decreased in the LPS + si-STIM1 group (Fig. 1G).

Figure 1 STIM1 expression is increased in LPS-induced A549 cells and STIM1 knockdown inhibits LPS-induced A549 cell apoptosis. The expression of STIM1 was detected by (A) RT-qPCR and (B) western blotting. The STIM1 interference plasmid was constructed and transfected into A549 cells and the transfection efficacy was detected by (C) RT-qPCR and (D) western blotting. (E) Cell viability was assessed by Cell Counting Kit-8. (F) Flow cytometry was used to detect cell apoptosis. (G) Western blotting was used to detect apoptosis-related proteins. **P<0.01 and ***P<0.001. STIM1, stromal interaction molecule 1; LPS, lipopolysaccharide; NC, negative control; RT-qPCR, reverse transcription-quantitative PCR; si, small interfering (RNA).

STIM1 knockdown alleviates LPS-induced inflammation and ERS in A549 cells

ELISA kits were used to detect the levels of inflammatory cytokines and the results indicated that LPS could significantly increase the levels of IL-6, IL-1β and TNF-α. Following inhibition of STIM1 expression, the levels of the inflammatory cytokines were reversed (Fig. 2A). An IF assay was used to detect the expression of CHOP and it was found that the fluorescence intensity of CHOP was significantly increased after LPS induction, while expression was significantly decreased after STIM1 inhibition (Fig. 2B). Western blotting analysis demonstrated that GRP78, ATF6, CHOP, caspase 12 and p-PERK expression levels were increased in the LPS group compared with the control group. Furthermore, compared with the LPS + si-NC group, the expression of the aforementioned proteins in the LPS + si-STIM1 group were decreased (Fig. 2C).

Figure 2 STIM1 knockdown alleviates LPS-induced A549 cell inflammation and ER stress. (A) Enzyme-linked immunosorbent assay kits were used to detect the levels of inflammatory cytokines. (B) Immunofluorescence assay was used to detect the expression of CHOP. (C) Western blotting was used to detect the expression of GRP78, ATF6, CHOP, caspase 12 and p-PERK. ***P<0.001. STIM1, stromal interaction molecule 1; LPS, lipopolysaccharide; ATF6, activating transcription factor 6; ER, endoplasmic reticulum; CHOP, C/EBP homologous protein; GRP78, glucose-regulated protein 78; NC, negative control; p-PERK, phosphorylated PKR-like ER kinase; si, small interfering RNA.

IGF2BP2 enhances the stability of STIM1 mRNA

The results of RT-qPCR and western blotting assays demonstrated that IGF2BP2 levels were also significantly increased in LPS-induced A549 cells (Fig. 3A and B). The binding ability of IGF2BP2 to STIM1 mRNA was detected using a RIP assay; as expected, STIM1 mRNA was enriched in RNA pulled down by anti-IGF2BP2 in cells (Fig. 3C). IGF2BP2 interference and overexpression plasmids were constructed and transfected into A549 cells and the transfection efficacy was detected by RT-qPCR and western blotting (Fig. 3D and E). Following this, si-IGF2BP2#1 was chosen for the follow-up experiments. After actinomycin D treatment, the stability of STIM1 mRNA decreased upon IGF2BP2 inhibition (Fig. 3F). Next, cells were divided into the control, si-NC, si-IGF2BP2, Oe-NC and Oe-IGF2BP2 groups and the STIM1 expression level in these groups was detected by RT-qPCR and western blotting. The results demonstrated that the expression of STIM1 decreased significantly after IGF2BP2 expression was inhibited. Furthermore, overexpression of IGF2BP2 significantly increased the expression of STIM1 in cells (Fig. 3G and H). Next, cells were divided into the control, LPS, LPS + si-STIM1, LPS + si-STIM1 + Oe-NC and LPS + si-STIM1 + Oe-IGF2BP2 groups. RT-qPCR results showed that STIM1 expression was significantly inhibited in LPS + si-STIM1 group compared with LPS group. Compared with LPS + si-STIM1 + Oe-NC group, the expression of STIM1 was increased in LPS + si-STIM1 + Oe-IGF2BP2 group (Fig. 3I).

Figure 3 IGF2BP2 enhances the stability of STIM1 mRNA. The expression of IGF2BP2 was detected by (A) RT-qPCR and (B) western blotting. (C) STIM1 mRNA levels were measured in RNA pulled down by anti-IGF2BP2 in cells. IGF2BP2 interference and overexpression plasmids were constructed and transfected into A549 cells and the transfection efficacy was detected by (D) RT-qPCR and (E) western blotting. (F) After actinomycin D treatment, the stability of STIM1 mRNA was detected by RT-qPCR. The STIM1 expression level was detected by (G) RT-qPCR and (H) western blotting. (I) The STIM1 expression level was detected by RT-qPCR after inhibiting STIM1 and overexpressing IGF2BP2 simultaneously. *P<0.05, ***P<0.001. IGF2BP2, insulin-like growth factor 2 mRNA binding protein 2; STIM1, stromal interaction molecule 1; RT-qPCR, reverse transcription-quantitative PCR; LPS, lipopolysaccharide; NC, negative control; Oe, overexpression; si, small interfering RNA.

Knockdown of IGF2BP2-regulated STIM1 expression alleviates LPS-induced ERS and inflammatory responses in A549 cells

The results demonstrated that overexpression of IGF2BP2 reversed the inhibitory effect of STIM1 knockdown on the LPS-induced apoptosis of A549 cells (Fig. 4A-C). The ELISA results demonstrated that, compared with the LPS + si-STIM1 + Oe-NC group, the levels of IL-1β and TNF-α in the LPS + si-STIM1 + Oe-IGF2BP2 group were significantly increased, IL-6 showed an increasing trend, but it was not marked (Fig. 4D). Furthermore, the results of the IF assay showed that overexpression of IGF2BP2 reversed the inhibitory effect of STIM1 knockdown on CHOP expression in LPS-induced A549 cells (Fig. 5A). In addition, western blotting analysis demonstrated that, compared with the LPS + si-STIM1 + Oe-NC group, the levels of GRP78, ATF6, CHOP, caspase 12 and p-PERK in the LPS + si-STIM1 + Oe-NC group were significantly increased (Fig. 5B).

Figure 4 Knockdown of IGF2BP2-regulated STIM1 expression alleviates LPS-induced inflammatory responses in A549 cells. (A) Cell viability was assessed by Cell Counting Kit-8. (B) Flow cytometry was used to detect cell apoptosis. (C) Western blotting was used to detect apoptosis-related proteins. (D) Enzyme-linked immunosorbent assay kits were used to detect the levels of inflammatory cytokines. *P<0.05 and ***P<0.001. IGF2BP2, insulin-like growth factor 2 mRNA binding protein 2; STIM1, stromal interaction molecule 1; LPS, lipopolysaccharide; NC, negative control; Oe, overexpression; si, small interfering RNA.

Figure 5 Knockdown of IGF2BP2-regulated STIM1 expression alleviates LPS-induced ER stress in A549 cells. (A) An immunofluorescence assay was used to detect the expression of CHOP. (B) Western blotting was used to detect the expression of GRP78, ATF6, CHOP, caspase 12 and p-PERK. **P<0.01, ***P<0.001. IGF2BP2, insulin-like growth factor 2 mRNA binding protein 2; STIM1, stromal interaction molecule 1; LPS, lipopolysaccharide; ER, endoplasmic reticulum; CHOP, C/EBP homologous protein; GRP78, glucose-regulated protein 78; ATF6, activating transcription factor 6; NC, negative control; Oe, overexpression; p-PERK, phosphorylated PKR-like ER kinase; si, small interfering (RNA).

Discussion

Pneumonia is a severe infectious disease attributed to a number of pathogenic factors, among which bacterial pneumonia is the most common. LPS, a major component of the gram-negative bacterial cell wall, is a common inducer of the inflammatory response in tissues and cells (26,27). After gram-negative bacteria enters the body, LPS can induce chemotaxis, migration and eventually infiltration of the lung tissues and, accompanied by the upregulation of inflammatory mediators, ultimately lead to the occurrence of acute lung injury (28). Hence, LPS is often used as an inducer for acute lung injury models. In the present study, LPS was used to construct a lung injury model using A549 cells. The results demonstrated that LPS led to significantly decreased cell viability and increased apoptosis and inflammatory response, which indicated the successful construction of the model.

Classical ERS receptors are ER membrane proteins, PERK and ATF6. Typically, when these receptor proteins are bound to the GRP78 molecular chaperone, they are inactive and become activated upon dissociation from GRP78 during ERS, triggering downstream events through signal transduction (29). In addition, upregulation of CHOP transcription factor may induce the inflammatory response and apoptosis and promote the occurrence and development of diseases (30,31). Inhibition of ERS-mediated apoptosis has been reported to reduce susceptibility to Streptococcus pneumoniae co-infection after influenza infection (32). Furthermore, ginsenoside Rg1 regulates sirtuin 1 and improves lung inflammation and injury caused by sepsis by inhibiting ERS and inflammation (33). In addition, caspase12 is a pro-apoptotic molecule specific to the outer membrane of the ER and activation of caspase12 is one of the core links in the development of ERS phase decay (34). In the present study, LPS was administered to A549 cells to induce ERS injury. The expression of ERS-related proteins, GRP78, ATF6, CHOP, caspase 12 and p-PERK, were significantly increased after LPS induction. These results indicated that LPS induced ERS injury in A549 cells.

In the present study, it was found that STIM1 expression was significantly elevated in LPS-induced A549 cells. STIM1 is a newly discovered human gene located in a specific region of human chromosome 11p15.5(35). STIM1 is involved in the regulation of the inflammatory processes. Regulation of STIM1/Orai1 signaling in bovine mammary epithelial cells has been shown to alleviate LPS-induced inflammation (17). In addition, the expression of STIM1 and Orai1 is upregulated when exposed to a high water volume or large cyclic stretching, which further activates calcium-sensitive protein kinase Cα, leading to calcium overload, excessive endothelial permeability and ultimately ventilator-induced lung injury (36). STIM1 activation also mediates S-phase stagnation and cell death in acute pulmonary poisoning induced by paraquat (37). In addition, STIM1-Orai1 interaction exacerbates LPS-induced bovine hepatocyte inflammation and ERS through store-operated calcium entry (9). However, to the best of the authors' knowledge, the role of STIM1 in LPS-induced A549 cell injury has not yet been reported. In the present study, it was found that inhibition of STIM1 expression in LPS-induced A549 cells significantly inhibited LPS-induced apoptosis, inflammation and ERS.

In the present study, through RIP and other experiments, it was demonstrated that IGF2BP2 could enhance the stability of STIM1 mRNA. A previous study demonstrated that IGF2BP2 knockdown inhibited LPS-induced inflammation of lung epithelial cells by targeting caspase 4, thus inhibiting the non-standard scorching pathway (19). In addition, ameliorating TGF-β-activated kinase 1 binding protein 3 N6-methyladenine modification by IGF2BP2-dependent mechanisms reduces kidney injury and inflammation (38). In the present study, it was found that overexpression of IGF2BP2 reversed the inhibitory effect of STIM1 knockdown on LPS-induced apoptosis, inflammation and ERS in A549 cells.

The present study also has certain limitations. First, it only investigated IGF2BP2-dependent STIM1 inhibition in cells and did not verify it in animals. The conclusions will be verified in animals in future experiments. Second, the present study showed that the abnormality of IGF2BP2 can affect the immune response of the body (39). Will the subsequent downregulation of IGF2BP2 indirectly affect the expression of STIM1, rather than because of the influence on STIM1? This will be explored further in future experiments. In addition, as for the experiments for STIM1 overexpression, it is still necessary to further verify the experiment and this will be discussed in future studies.

In conclusion, knockdown of IGF2BP2-regulated STIM1 expression protects against LPS-induced pneumonia in vitro by alleviating ERS and the inflammatory response.

Supplementary Material

Supplementary Data

Acknowledgements

Not applicable.

Funding

Funding: The present study was supported by National K&D Program of China (grant no. 2022YFC2304800), Guangzhou Medical Key Discipline (grant no. 2021-2023) and Guangzhou Science and Technology Planning Project (grant no. 2023A03J0534).

Availability of data and materials

The data sets analyzed and/or generated during the present study are available from the corresponding author on reasonable request.

Authors' contributions

JH conceived the present study. WZ, QD, NS and ZL performed the experiments. WZ wrote the manuscript. QD, NS and ZL processed the experimental data and JH ensured the accuracy of the experimental data. WZ and JH confirm the authenticity of all the raw data. All authors read and approved the final manuscript.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References