The alveolar epithelial cell line MLE-12 was purchased from the BeNa Culture Collection; Beijing Beina Chunglian Institute of Biotechnology. Cells were cultured in DMEM/F12 Coon's medium (DMEM/F12; Thermo Fisher Scientific, Inc.) containing 10% FBS (Thermo Fisher Scientific, Inc.) and maintained in a 5% CO₂ incubator at 37˚C. After the cells were passaged three times, cells in logarithmic phase were selected for follow-up assays.

The sepsis cell model was established using MLE-12 cells. Cells in the logarithmic phase were routinely cultured for 24 h at 37˚C. When the degree of cell fusion reached 90%, 100 ng/ml LPS (Sigma-Aldrich; Merck KGaA) was added to the culture for 24 h at 37˚C based on data from a previous study (18).

Microarray data were obtained from the GEO database (www.ncbi.nlm.nih.gov/geo). Gene expression profiles of the neonatal sepsis samples public dataset GSE145227 was downloaded from GEO (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi) (19). The GSE145227 dataset contained 10 samples from children with sepsis and 12 samples from healthy controls. Affymetrix Human lncRNA Array (version 1.0; Affymetrix; Thermo Fisher Scientific, Inc.) was used to detect the expression levels of long non-coding (lnc)RNAs and mRNAs in both septic and control groups. Subsequently, the data were analyzed using GEO2R (https://www.ncbi.nlm.nih.gov/geo/geo2r) and GraphPad Prism 8.0 software (GraphPad Software, Inc.). Search tool for recurring instances of neighboring genes (STRING) database (version 11.0; https://version-11-0.string-db.org/) was used to analyze the potential association between STOM and CD36.

Small interfering si(RNA) against STOM (si-STOM; si-STOM-1, 1 µg, 5'-GTGTTTCTAAAGATGGAATTTCA-3'; si-STOM-2, 1 µg, 5'-GAGTCATCTATTCTGATTATTTG-3') and the scrambled negative control group (si-NC; 1 µg; 5'-ACTTGCGCTTGCGAAAATCTATATAGC-3') were constructed by Guangzhou RiboBio Co., Ltd. The CD36-overexpression vector (Ov-CD36; 50 nM) and empty control vector (Ov-NC; 50 nM) were constructed by Shanghai GenePharma Co., Ltd. siRNA and plasmids were transfected into MLE-12 cells were inoculated into six-well plates (5x10⁵ cells/well), and then transfection was performed for 8 h at 37˚C using Lipofectamine^® 2000 (Thermo Fisher Scientific, Inc.). After transfection for 48 h, the transfection efficiency of cells in each group was detected via reverse transcription-quantitative (RT-q)PCR. Subsequent experiments were completed within 48 h.

TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) was used to extract total RNA from cells. Following which, 5 µg total RNA was used as the control and cDNA was synthesized using AMV reverse transcription kit (Promega Corporation) according to the manufacturer's protocol. qPCR was performed on an ABI Prism 7900 Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.) using a SYBR^® Green kit (Qiagen, Inc.). The relative expression level of the target gene was calculated using the 2^-ΔΔCq method (20). GAPDH was used for normalization. The following thermocycling conditions were used: Initial denaturation at 95˚C for 10 min; followed by 37 cycles of denaturation at 95˚C for 15 sec and annealing at 60˚C for 1 min; extension for 10 min at 65˚C. The sequences of PCR primers were as follows: CD36 Forward, 5'-AAGCCAGGTATTGCAGTTCTTT-3' and reverse, 5'-GCATTTGCTGATGTCTAGCACA-3'; STOM forward, 5'-GTGACTCTCGACAATGTAAC-3' and reverse, 5'-TGATCTCATAACGGAGGCAG-3'; and GAPDH forward, 5'-TGTCAAGCTCATTTCCTGGTAT-3' and reverse, 5'-CTCTCTTCCTCTTGTGCTCTTG-3'.

Transfected cells were centrifuged at 1,000 x g for 5 min at 4˚C. The supernatant was collected and used for ELISA. The levels of malondialdehyde (MDA), superoxide dismutase (SOD), lactate dehydrogenase (LDH), TNF-α and ROS in the cell supernatant were measured using the MDA assay kit (cat. no. A003-1-2; Nanjing Jiancheng Bioengineering Institute), SOD Assay Kit (cat. no. A001-3-2; Nanjing Jiancheng Bioengineering Institute), LDH Cytotoxicity Assay Kit (cat. no. C0016; Beyotime Institute of Biotechnology), TNF-α assay kit (cat. no. H052-1; Nanjing Jiancheng Bioengineering Institute) and ROS assay kit (cat. no. S0033S; Beyotime Institute of Biotechnology), respectively, according to the manufacturer's protocol. These samples were measured using a Hitachi spectrophotometer (F-7000 FL spectrophotometer; Hitachi, Ltd.).

Cells were inoculated on 96-well plates at a density of 4x10⁴ cells/well. The cells were cultured in DMEM/F12 medium containing 10% FBS for 24, 48 and 72 h at 37˚C. Subsequently, 10 µl CCK-8 solution (cat. no. C0038; Beyotime Institute of Biotechnology) was added to each well to culture for 1 h. The absorbance of cells in each well was detected at 450 nm using an automatic enzyme labeling instrument.

The RIP assay was performed using the Magna RIP RNA-Binding Protein Immunoprecipitation kit (including RIP buffer and beads; cat. no. 17-700; MilliporeSigma) according to the manufacturer's protocol. Cells were incubated with RIP buffer supplied with the kit in an ice bath for 20 min. A total of 20 µl magnetic beads were applied for incubation of cell extracts and coated with 1 µg Ago2 antibody (cat. no. 03-110; Merck KGaA) or 2 µg IgG antibody supplied with the kit for 12 h at 40˚C. Immunoprecipitated complexes were collected by adding 500 µl lysate per 200 µl RIP buffer, followed by centrifugation at 10,000 x g for 10 min at 4˚C. and RT-qPCR was subsequently performed.

Total protein was extracted from MLE-12 cells using RIPA lysis buffer (Beyotime Institute of Biotechnology). The protein concentration was detected using a BCA protein quantitative kit (Pierce; Thermo Fisher Scientific, Inc.). A total of 25 µg protein per lane was separated via SDS-PAGE on a 10% gel, and subsequently transferred to PVDF membranes. These membranes were blocked with 5% non-fat milk for 2 h at room temperature. Membranes were incubated with primary antibodies (1:1,000) against STOM (cat. no. ab166623), CD36 (cat. no. ab133625), TNF-α (cat. no. ab215188), IL-6 (cat. no. ab233706) and GAPDH (cat. no. ab9485) (all Abcam) overnight at 4˚C. After the membranes were washed with TBST (0.1% Tween-20) three times, membranes were incubated with HRP-conjugated goat anti-rabbit (1:2,000; cat. no. 14708; Cell Signaling Technology, Inc.) secondary antibody for 2 h at room temperature. ECL reagent (Pierce; Thermo Fisher Scientific, Inc.) was used to detect the protein bands. ImageJ software 1.4.3 (National Institutes of Health) was used to detect the relative gray value of the target proteins.

GraphPad Prism 8.0 software (GraphPad Software, Inc.) was used for statistical analysis. The data are expressed as the mean ± standard deviation (unless otherwise shown). One-way ANOVA followed by Tukey's post hoc test was used to analyze the comparison between multiple groups. An unpaired Student's t-test was used to compare differences between two groups. P<0.05 was considered to indicate a statistically significant difference.

GEO2R analysis of the GeneChip GSE145227 was employed to confirm that the expression level of STOM was significantly increased in the peripheral blood of newborns with sepsis compared with in the peripheral blood of healthy newborns (Fig. 1A). Subsequently, the results of RT-qPCR and western blotting demonstrated that the expression level of STOM in the sepsis model group was significantly higher compared with the control group (Fig. 1B and C). These results suggested that STOM may be involved in LPS-induced damage of MLE-12 cells.

The expression levels of STOM in MLE-12 cells were decreased following transfection with si-STOM. As demonstrated in Fig. 1D and E, the expression levels of STOM in the si-NC group were similar to the control group; whereas STOM expression was decreased in both the si-STOM-1 and si-STOM-2 groups compared with the control group, with si-STOM-1 exhibiting an increased level of transfection efficiency compared with si-STOM-2. Therefore, si-STOM-1 was selected for follow-up experiments. A CCK-8 assay was applied to detect cell viability. As presented in Fig. 1F, the results demonstrated that treatment with LPS significantly inhibited cell viability compared with the control group, while STOM-knockdown reduced the inhibitory effect of LPS on cell viability compared with LPS+si-NC group.

Oxidative stress and inflammation levels were detected using ELISA and western blotting assays. As demonstrated in Fig. 2A-D, compared with the LPS + si-NC group, the levels of LDH, MDA and ROS in the LPS + si-STOM group were significantly decreased, whereas the level of SOD was significantly increased. Moreover, treatment with LPS induced high levels of LDH, MDA and ROS, and low levels of SOD compared with the control group. The results of western blotting and RT-qPCR analyses demonstrated that compared with the control group, LPS significantly increased the expression levels of the pro-inflammatory cytokines TNF-α and IL-6, whereas these changes were partially reversed by STOM-knockdown (Fig. 2E). Similar results were observed using ELISA. As presented in Fig. 2F, results of ELISA revealed that compared with the LPS + si-NC group, STOM-knockdown significantly decreased the levels of the pro-inflammatory cytokines TNF-α and IL-6. These results indicated that STOM-knockdown reduced oxidative stress and inflammation in MLE-12 cells treated with LPS.

To further explore the effects of STOM on the damage of MLE-12 cells treated with LPS, the downstream target gene of STOM, CD36, was screened for using the STRING database (Fig. 3A). The interaction between STOM and CD36 was verified using a RIP assay (Fig. 3C). The results revealed that CD36 and STOM bound strongly. Moreover, as demonstrated in Fig. 3B, STOM-knockdown significantly decreased the expression of CD36 compared with the control. Thus, results of the present study demonstrated that STOM positively regulated CD36.

Lastly, a CD36-overexpressing plasmid (Ov-CD36) was constructed. As displayed in Fig. 4A and B, compared with the Ov-NC group, both the protein and mRNA expression levels of CD36 were significantly increased in the Ov-CD36 group. Detection of oxidative stress levels using the kits revealed a significant decrease in LDH levels after interfering with STOM compared with the LPS + si-NC group. However, the inhibitory effect of interfering with STOM on LDH was reversed after CD36 overexpression (Fig. 4C). Additionally, the trend of MDA and ROS among groups was similar to that of LDH (Fig. 4D and F). By contrast, following interference with STOM, SOD levels were upregulated compared with the LPS + si-NC group, but following CD36 overexpression, SOD levels were decreased (LPS + si-STOM + Ov-CD36 vs. LPS + si-STOM + Ov-NC; Fig. 4E). As demonstrated in Fig. 4C-F, the overexpression of CD36 reversed the inhibitory effect of STOM-knockdown on oxidative stress. As demonstrated in Fig. 4G and H, TNF-α and IL-6 expression was suppressed after knockdown of STOM compared with the LPS + si-NC group. However, overexpression of CD36 significantly increased TNF-α and IL-6 expression compared with the knockdown of STOM group. Therefore, it was clear that overexpression of CD36 alleviated the inhibitory effect of STOM-knockdown on inflammation. Collectively, these results demonstrated that STOM aggravated LPS-induced damage of MLE-12 cells by promoting the expression of CD36.