The miR-214-3p/CTSD Axis Regulates Lysosomal Homeostasis in Porcine Intestinal Epithelial Cells: A Preliminary Study

Lysosomes are crucial for the function of fetal vacuolated enterocytes in neonatal piglets, yet how they are regulated by miRNAs remains poorly defined. Therefore, this study aimed to elucidate how miRNAs govern lysosomal homeostasis in the developing intestine. Using a neonatal piglet model of lysosomal dysfunction induced by imipramine (IMI), we identified ssc-miR-214-3p as a key down-regulated miRNA implicated in lysosomal pathways. In IPEC-J2 enterocytes, the miR-214-3p mimic ameliorated IMI cytotoxicity by restoring cell viability and migration while suppressing apoptosis. Further analysis revealed that miR-214-3p directly reversed the lysosomal defects triggered by IMI treatment. Specifically, it alleviated lysosomal alkalinization and markedly restored acid phosphatase (ACP) activity, indicating a recovery of the acidic hydrolytic environment. This restoration was also accompanied by the preservation of lysosomal membrane integrity and a consequent reduction in the nuclear translocation of transcription factor EB (TFEB). Furthermore, cathepsin D (CTSD) was validated as a direct target of miR-214-3p by luciferase assay, and its overexpression reversed the protective effects of the mimic on lysosomal acidification and lysosome-associated membrane protein 1 (LAMP1) levels. Collectively, our findings reveal a novel miR-214-3p/CTSD axis that regulates lysosomal homeostasis during neonatal intestinal maturation, providing a potential therapeutic target for porcine intestinal disorders.