Integrated Bioinformatics Analysis and Experimental Validation Reveal Immune-Related miRNA–mRNA Networks in Tuberculosis

Acquisition and processing of public tuberculosis-related expression datasets1516 171822Functional enrichment analysis of DE-mRNAsTo explore the biological significance of the DE-mRNAs, Gene Ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed using the “clusterProfiler” and “enrichplot” packages in R. Functional terms related to immune response, cytokine activity, and infection pathways were prioritized. A false discovery rate (FDR) <0.05 was considered statistically significant.miRNA target prediction and regulatory network constructionThe targeted genes of DE-miRNAs were predicted using online tool miRNet2.0 with a cumulative weighted context++ score greater than -0.5. These predicted targets were intersected with DE-mRNAs to identify negatively correlated miRNA-mRNA pairs. Cytoscape (version 3.9.1) was used to construct and visualize the miRNA-mRNA regulatory network.MTB infected cell model, sequencing and data analysis25A small RNA (sRNA) library was constructed from 100 ng of total RNA per sample using QIAseq miRNA Library Kit (Life Technologies, USA) and sequencing was performed using Illumina novaseq 6000 (Illumina, USA). Raw data were filtered using fastp (version 0.23.1) and aligned to the reference genome using Bowtie software. Known miRNAs were identified by alignment to miRBase, with secondary structure and expression profiles analyzed using mirdeep2 and srna-tools-cli. DE-miRNAs were analyzed using DESeq2. miRNA targets were predicted using miRanda and RNAhybrid, with consensus results used to construct regulatory networks. KEGG pathway enrichment was performed using “clusterProfiler” package.Reverse transcription quantitative PCR–ΔΔCtThis study was conducted in accordance with the Declaration of Helsinki and approved by the local Institutional Review Board (NO.2021JS010). Written informed consent was obtained from all participants prior to sample collection. All personal identifiers were removed to ensure participant anonymity and data confidentiality.ELISASerum samples and cell supernatants were collected, and ELISA was performed to detect levels of cytokines interferon-gamma (IFN-γ), interleukin (IL)-1β, IL-2, IL-6, IL-10, and tumor necrosis factor-alpha (TNF-α) according to the manufacturer’s protocol (RX106205H; Ruixin Biotech., Quanzhou, China). Dual-luciferase reporter assayTo validate miRNA-target interactions, wild-type (WT) and mutant (MUT) 3′-untranslated regions (3′-UTRs) of IFITM1 and SAMD9L containing predicted miRNA-binding sites were cloned into the GP-miRGLO dual-luciferase vector (GenePharma, Shanghai, China). HEK-293T cells (5×10⁴ cells/well in 24-well plates) were co-transfected with WT or MUT reporter plasmid, miRNA mimic, or negative control (NC), and pRL-TK Renilla luciferase plasmid (GenePharma) as an internal control using GP-transfect-Mate (GenePharma) according to the manufacture’s protocol. After a 48-hour incubation, luciferase activity was measured using a dual-luciferase reporter assay kit (GenePharma). miRNA mimic transfectionTHP-1 cells were transfected with miRNA mimics (hsa-miR-130a-3p and hsa-miR-146a-5p, Table 2) from Chinese Academy of Sciences using RNATransMate (Sangon, Shanghai, China) according to the manufacturer’s instructions. At 24 or 48 hours post-transfection (LC3, P62 and Caspase 3), the cells were infected with BCG and incubated at 37°C for another 24 hours. Cells and supernatants were harvested. Levels of apoptosis- and autophagy-related proteins in cells were assessed via western blot. Supernatant cytokine levels were quantified using ELISA kits (Jingmei, Shenzhen, China), following the manufacturer's protocols.Western blotCells were lysed using RIPA buffer (Solarbio, Peking, China) containing proteinase/phosphatase inhibitor (Baisha, China) at 4°C for 30 min. Protein samples were prepared, and their concentrations were determined using a bicinchoninic acid (BCA) protein assay kit. (Mmouse, Xian, China). Samples were separated on a 10% or 15% polyacrylamide gel by electrophoresis at 80 V (constant voltage) for 1 h. The proteins were transferred to a PVDF membrane at a constant current of 250 mA for 2 hours. After the transfer, the membrane was incubated in a 5% blocking solution (prepared with Tris-Buffered Saline with Tween 20 (TBST) and skim milk) for 1 h at room temperature. Membranes were blocked with 5% BSA in TBST for 2 h at room temperature and incubated overnight at 4°C with the following primary antibodies: Beclin1 (PB9076; Boster, Wuhan, China), LC3B (12714S; CST, Boston, USA), p62 (18420; Proteintech, Chicago, USA), and Casspase3 (T40044F; Abart, Shanghai, China) (all 1:1000 diluted in blocking buffer). The membrane was then incubated for 2 h at room temperature with HRP-conjugated secondary antibodies (1:5000 dilution in TBST). Signals were detected using a freshly prepared enhanced chemiluminescence (ECL) working solution (Biosharp, Anhui, China) and imaged with a cooled CCD camera. The image was processed and analyzed using ImageJ (version 2.2.0-beta6). Apoptosis by flow cytometryCells were harvested and suspended in ice-cold PBS. After discarding the supernatant, cells were stained with Annexin V-FITC and propidium iodide (PI) for 20 min at room temperature in the dark according to the manufacturer’s protocol. Then cells were subjected to flow cytometry using a CytoFLEX flow cytometer (Beckman, Breu CA, USA).