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.