Research Paper Volume 12, Issue 18 pp 18588—18602

Lipopolysaccharide upregulates miR-132/212 in Hirschsprung-associated enterocolitis, facilitating pyroptosis by activating NLRP3 inflammasome via targeting Sirtuin 1 (SIRT1)

Figure 5. Upregulation of miR-132/212 triggers pyroptosis in HT29 cells. (A and B) qRT-PCR and western blot analysis of NLRP3, Caspase-1 and ASC were detected in HT29 cell line after transfection with miR-132/212 mimics (or Con mimics). Mean ± SD. n = 3, *P < 0.05. (C) Western blot analysis showed that the protein level of SIRT1 was up-regulated in HT29 cell line exposed to 1000 ng/ml LPS for 24h immediately after transfection with miR-132/212 inhibitor, while NLRP3, Caspase-1 and ASC protein levels were decreased. Data are means ± SEs of three independent experiments. (D) Confocal images were performed to determine the staining intensity of SIRT1 and NLRP3 in HT29 cell line treated with 1000 ng/ml LPS for 24h after transfection with miR-132/212 inhibitor (or Con inhibitor). Scale bar =25μm. (E) Flow cytometric analysis using active caspase-1 marker was examined in HT29 cell line treated with no or 1000 ng/ml LPS for 24h. MiR-132/212 inhibitor and SIRT1 OE transfection were separately done in HT29 cell line, and then HT29 cell line was exposed to 1000 ng/ml LPS for 24h. Con, control; OE, overexpression.