MiR-125b-5p targets Bak1 to regulate HCT-8 cell apoptosis in response to Cryptosporidium parvum infection via mitochondrial pathway.

BACKGROUND: Cryptosporidium parvum infection can regulate apoptosis of host epithelial cells through both extrinsic and intrinsic apoptotic pathways, potentially involving various microRNAs (miRNAs). This study focuses on the role of miR-125b-5p in modulating apoptosis in HCT-8 cells in response to C. parvum infection. METHODS: Expression levels of miR-125b-5p and C. parvum burden were estimated using real-time quantitative polymerase chain reaction (RT-qPCR) and cell apoptosis was detected by flow cytometry. The interaction between miR-125b-5p and Bak1 was studied by luciferase reporter assay, RT-qPCR, and western blotting. The expression of Cytochrome c (Cyt c), Caspase-3, and Caspase-9 were assessed by RT-qPCR, and western blotting. JC-1 assess mitochondrial membrane potential. RESULTS: MiR-125b-5p expression decreased at 3, 6, 12, and 24 h post-infection (hpi) but increased at 48 and 72 hpi. Upregulation of miR-125b-5p inhibited apoptosis and increased parasite burden in HCT-8 cells following C. parvum infection, whereas its downregulation promoted apoptosis and reduced the parasite burden. Luciferase reporter assays identified Bak1 as a target gene of miR-125b-5p. Suppression of Bak1 expression via small interfering RNA (siRNA) inhibited apoptosis and increased the C. parvum burden, while Bak1 overexpression promoted apoptosis and reduced the parasite burden. Co-transfection with miR-125b-5p mimics and pcDNA3.1-Bak1 demonstrated that miR-125b-5p regulates apoptosis and parasite burden by targeting Bak1. Further analysis showed that overexpression of miR-125b-5p significantly decreased the mRNA and protein levels of Cytochrome c (cyt-c), caspase-3, and caspase-9, as well as reduced mitochondrial membrane damage. Inhibition of miR-125b-5p produced opposite effects. Co-transfection with miR-125b-5p mimics and pcDNA3.1-Bak1 revealed that pcDNA3.1-Bak1 counteracted the inhibitory effects of miR-125b-5p on cyt-c, caspase-3, caspase-9, and mitochondrial membrane damage. CONCLUSIONS: The present data indicated that miR-125b-5p targets Bak1 to regulate apoptosis in HCT-8 cells in response to C. parvum infection via the mitochondrial pathway.