Background <p>Gastric cancer remains one of the leading causes of cancer-related death worldwide. miR-214-3p, a microRNA, has been reported to exhibit dysregulated expression and play important regulatory roles in various cancers. However, the global targets and underlying mechanisms of miR-214-3p in gastric cancer progression remain poorly understood.</p> Methods <p>In this study, we performed long-read transcriptomic sequencing to comprehensively characterize transcriptome-wide variations in AGS gastric cancer cells following miR-214-3p overexpression (OE) or knockdown (KD). Functional in vitro experiments were conducted to validate the newly identified transcripts and assess the effects of miR-214-3p OE on cell proliferation and apoptosis in AGS cells.</p> Results <p>Our results demonstrated that miR-214-3p OE and KD significantly altered the expression levels of numerous genes at both transcript and gene levels, particularly those involved in apoptosis and transcriptional regulation. Interestingly, miR-214-3p KD and OE showed consistent directional effects on the expression of some target genes. Additionally, miR-214-3p broadly influenced the alternative splicing of hundreds of genes, including those associated with cell division, senescence, and transcriptional regulation. Notably, changes in miR-214-3p expression also affected the alternative polyadenylation and 3’UTR lengths of transcripts. Furthermore, our in vitro experiments revealed that miR-214-3p OE significantly promoted the proliferation of AGS cells and inhibited apoptosis, suggesting its potential oncogenic role.</p> Conclusions <p>Collectively, these findings highlight the potential multifaceted regulatory roles of miR-214-3p in mediating proliferation and apoptosis through diverse mechanisms in AGS gastric cancer cells, thereby advancing our understanding of its critical role in gastric cancer progression and its therapeutic potential.</p>