Integrated bioinformatics analyses and cell line experiments implicate miR-33a-5p/HIF1A in the cellular response to...

Celastrol has reported antitumor effects in lung cancer, but database-derived target lists and pathway predictions vary with the filters used. We examined which findings remained supported after evidence review and then tested the leading miRNA-target relationship in lung adenocarcinoma (LUAD) cells. Celastrol-associated proteins were curated from CTD and ChEMBL. Candidate miRNAs were reassessed by a reproducible literature search and an independent TCGA-LUAD screen. Alternative miRNA-target and disease-expression rules were compared. Network, enrichment, prognosis, monotherapy sensitivity, and structure analyses were retained as secondary analyses. A549 and H1975 cells were used for viability, qRT-PCR, HIF1A 3'UTR reporter, and miR-33a-5p inhibitor-rescue assays. Twenty-three proteins met the activity-based target rule; five had a retained direct binding measurement. The strict target/miRNA-target/LUAD differential-expression intersection was empty. Under an FDR-only disease rule, HIF1A was retained by four of five miRNA-target definitions but not by the ≥ 2-source rule. HIF1A expression was higher in GSE10072 LUAD tumors than in normal lung (log2 fold change = 0.566; Wilcoxon P = 4.24 × 10^-9). Celastrol increased miR-33a-5p and reduced HIF1A mRNA in both cell lines. Mutation of the tested HIF1A 3'UTR site attenuated the response to miR-33a-5p mimic, with significant reporter-by-mimic interactions in A549 (P = 2.41 × 10^-5) and H1975 cells (P = 6.31 × 10^-4). miR-33a-5p inhibition partly restored HIF1A mRNA and viability during celastrol treatment. HSP90 transcript results were inconsistent between cell lines. The prognostic model showed modest internal validation, and CellMiner results represented monotherapy patterns only. The cell line data support miR-33a-5p/HIF1A as a candidate contributor to the response to celastrol. Its computational selection depended on the disease and target filters. HSP90, docking, prognosis, and drug-screening findings require separate validation.