ABSTRACT: This study employed H9C2 cells (a rat myocardial cell line) as the experimental model, with three experimental groups and four biological replicates per group, resulting in a total of 12 samples. The experimental design aimed to investigate the regulatory role of LITAF in H9C2 cells under hypoxia reoxygenation (H/R) injury conditions. Specifically, the control group consisted of H9C2 cells cultured under normal conditions without H/R treatment or lentiviral transfection. The hypoxia reoxygenation (H/R) group consisted of H9C2 cells transfected with an empty lentiviral vector as a control, followed by exposure to hypoxia (1 percent O2) for 8 hours and subsequent reoxygenation under normoxic conditions for 2 hours. The hypoxia reoxygenation plus LITAF overexpression group consisted of H9C2 cells transfected with a LITAF overexpressing lentiviral vector to achieve LITAF overexpression, followed by hypoxia (1 percent O2 for 8 hours) and reoxygenation (2 hours) under the same conditions as the H/R group. After completion of the treatments, total RNA was extracted from each sample using a standard RNA extraction kit, and RNA quality was assessed, including RNA integrity number (RIN) evaluation, to ensure suitability for sequencing. RNA sequencing libraries were constructed according to standard protocols. Eukaryotic mRNA was enriched using Oligo(dT) magnetic beads, fragmented into approximately 300 bp fragments, reverse transcribed into cDNA, ligated with sequencing adapters, size selected, PCR amplified, and purified to obtain the final libraries. High throughput sequencing was performed on the Illumina NovaSeq X Plus platform using a paired end 150 bp sequencing strategy. Each sample was sequenced to a depth of approximately 6 to 10 Gb to ensure sufficient coverage for downstream analyses. Bioinformatics analyses included raw data quality control, alignment of sequencing reads to the rat reference genome, gene expression quantification, identification of differentially expressed genes using DESeq2 and/or edgeR, functional annotation using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases, and functional enrichment analyses. By comparing the transcriptomic profiles of the H/R group and the H/R plus LITAF overexpression group, with the control group serving as a baseline reference, this study aimed to identify LITAF-regulated differentially expressed genes, characterize enriched signaling pathways, and elucidate the molecular mechanisms by which LITAF modulates H/R-induced injury in H9C2 myocardial cells.
