Objective To study the molecular characteristics of RNA binding protein aldehyde dehydrogenase 18 family member A1 (ALDH18A1) in esophageal carcinoma cells (KYSE150 cells) and its effect on tumor growth. MethodsHuman esophageal squamous cell (KYSE150 cells) was cultured in vitro. At the same time, RNA co-immuno precipitation technology was used to study the binding of RNA and protein in the cell, and the corresponding RNA-protein complex was precipitated by the antibody of the target protein to separate and purify the captured RNA. The molecular characteristics of ALDH18A1 binding RNA were analyzed, and KyotoEncyclopedia of Genes and Genomes cluster analysis was performed for ALDH18A1 binding target genes. Results Protein immunoblotting experiments showed that the target protein was well enriched by antibodies. ALDH18A1 had extensive RNA binding activity, with significant enrichment in regions such as coding sequences, intron, and 5’untranslated region. ALDH18A1 mainly bound to the UGUAAUC motif of RNA. The cluster analysis showed that the RNA molecules bound to ALDH18A1 mainly participated in focal adhesion, central carbon metabolism in cancer, cell cycle, spliceosome, RNA transport, and ubiquitin mediated protein hydrolysis. Conclusion ALDH18A1 has the function of binding to RNA molecules and may play a role in the expression of esophageal cancer-related genes and related biological processes.
Objective To investigate the inhibitory effects and related mechanisms of NOD like receptor protein 3 (NLRP-3) inflammasome inhibitor MCC950 on oxidative stress, inflammation, and pyroptosis in human esophageal epithelial cells (HEECs). MethodsHEECs cells were passaged and divided into blank control group, acid stimulation group (stimulated 3 times a day with pH 4 acidic medium for 15 minutes each time, cultured for 48 hours), bile salt stimulation group (stimulated 3 times a day with 400 μmol/L bile salt mixture for 15 minutes each time, cultured for 48 hours), lipopolysaccharide (LPS) group (stimulated with 10 μL of 100 ng/mL LPS for 48 hours), MCC950 group (stimulated HEECs cells with 10 μL of 7.5 ng/mL MCC950 for 4 hours, then stimulated with acid, bile hydrochloric acid, and LPS for 48 hours), and N-acetyl-L-cysteine (NAC) group (stimulated HEECs cells with 1 mmol/L NAC for 4 hours, then stimulated with acid, bile hydrochloric acid, and LPS and incubated for 48 hours). Three culture dishes were used in each group to detect the mRNA and protein expression levels of oxidative protein/antioxidant protein [Nox-4 (NADPH oxidase 4), nuclearfactor erythroidderived 2-like 2 (Nrf-2), heme oxygenase-1 (HO-1)], NLRP-3 signaling pathway [NLRP3/caspase-1/intereukin-1β (IL-1β)/intereukin-18 (IL-18)], and cell apoptosis pathway [caspase-4/caspase-5/GSDMD] using real-time reverse transcription polymerase chain reaction (RT-PCR) and Western blotting experiments. Cell apoptosis were observed through Hoechst 33342 staining. ResultsMCC950 intervention (average optical density: 0.023) and NAC intervention (average optical density: 0.031) effectively inhibited HEECs apoptosis induced by acid (average optical density: 0.042), bile salt (average optical density: 0.047), and LPS (average optical density: 0.054). The results of RT-PCR and Western blotting experiments showed that MCC950 intervention and NAC intervention significantly inhibited the high expression of Nox-4 mRNA (MCC950:1.68±0.18, NAC: 1.62±0.17) and protein in HEECs cells induced by acid (1.00±0.05), bile salt (3.07±0.25), and LPS (3.52±0.37); And significantly upregulated the mRNA and protein expression levels of antioxidant proteins Nrf-2 (MCC950: 0.72±0.12, NAC: 0.57±0.12) and HO-1 (MCC950: 0.74±0.12, NAC: 0.57±0.12). MCC950 intervention and antioxidant NAC intervention effectively inhibited the mRNA and protein expression levels of acid stimulated, bile salt stimulated, and LPS induced HEECs cell NLRP-3 (MCC950 intervention: 1.58±0.06, NAC intervention: 1.47±0.09), ASC (MCC950 intervention: 1.56±0.09, NAC intervention: 1.93±0.17), caspase-1 (MCC950 intervention: 1.64± 0.13, NAC intervention: 1.96±0.20), IL-1β (MCC950 intervention: 1.66±0.18, NAC intervention: 1.82±0.20), IL-18 (MCC950 intervention: 1.58±0.13, NAC intervention: 1.84±0.16) and other indicators. MCC950 intervention and antioxidant NAC intervention effectively inhibited the mRNA and protein expression levels of apoptosis pathway markers such as caspase-4 (MCC950 intervention:1.51±0.03, NAC intervention: 1.61±0.12), caspase-5(MCC950 intervention: 1.38±0.13, NAC intervention: 1.64±0.21), and GSDMD (MCC950 intervention: 1.41±0.04, NAC intervention: 1.54±0.10) induced by acid stimulation, bile salt stimulation, and LPS in HEECs cells. ConclusionAcid, bile salts, and LPS can all induce the overexpression of oxidative stress markers in HEECs, reduce the expression of antioxidant proteins, and activate the NLRP3 inflammasome signaling pathway and cell pyroptosis pathway, promoting cellular inflammatory damage, but MCC950 has a protective effect.