Fig. 2: MiR-199a-3p regulates autophagy and apoptosis in cells to limit the C.

parvum load after infection. A Expression levels of miR-199a-3p in HCT-8 cells at different times after C. parvum sporozoites infection, determined with RT-qPCR (n  =  3 biologically independent samples). B Expression efficiency of miR-199a-3p in HCT-8 cells transfected with miR-199a-3p mimic or miR-199a-3p inhibitor for 24 h, determined with RT-qPCR. C Effect of miR-199a-3p on autophagy of cells infected with C. parvum. Cells were transfected with miR-199a-3p mimic or miR-199a-3p inhibitor for 24 h and then exposed to equal numbers of C. parvum sporozoites for 12 h. Expression levels of autophagy-related proteins LC3, P62, and Beclin 1 were determined with western blotting. Protein levels of LC3-II, P62, and Beclin 1 relative to GAPDH levels were determined with densitometry(n  =  3 biologically independent samples). D Effect of miR-199a-3p on autophagic flux in C. parvum-infected cells. Cells were cotransfected with miR-199a-3p mimic or miR-199a-3p inhibitor and a plasmid encoding mCherry–EGFP–LC3 for 24 h, and then exposed to equal numbers of C. parvum sporozoites for 12 h (n = 3 biologically independent samples). The autophagic flux was evaluated with confocal microscopy. Numbers of yellow and free red dots in each group cell were quantified (free red dots: autophagic lysosomes; yellow dots: autophagosome, Scale bar, 5 µm). E Effect of miR-199a-3p on apoptosis of cells infected with C. parvum. Cells were transfected with miR-199a-3p mimic or miR-199a-3p inhibitor for 24 h and then exposed to equal numbers of C. parvum sporozoites for 12 h. Cell apoptosis was detected with flow cytometry. The apoptosis rate is the sum of O1-LR and O1-UR (n  =  3 biologically independent samples). F Cells were transfected with miR-199a-3p mimic or miR-199a-3p inhibitor for 24 h and then exposed to equal numbers of C. parvum sporozoites for 2 h to determine the initial attachment and cellular invasion of C. parvum with RT-qPCR. The medium was replaced with fresh medium at 3 hpi infection with C. parvum and the infected cells cultured for a further 12 or 24 h to evaluate the parasite burden after invasion(n  =  3 biologically independent samples). All data presented are the means ± SD of three independent experiments. Different groups were compared with a t test (*P < 0.05, **P < 0.01, ***P < 0.001).