NLRP12 promotes host resistance against Pseudomonas aeruginosa keratitis inflammatory responses through the negative regulation of NF-κB signaling.

OBJECTIVE: To investigate the role of NLRP12 in regulating Pseudomonas aeruginosa (P. aeruginosa) keratitis. MATERIALS AND METHODS: Real-Time-PCR and Western blot were performed to measure the NLRP12 level in corneas and bone marrow-derived macrophages (BMDMs) of C57BL/6 (B6) mice. B6 mice received a subconjunctival injection of lentivirus expressing active NLRP12 (NLRP12-lentivirus) or Ctl-lentivirus (as control), followed by infection of P. aeruginosa. The clinical score, slit lamp and bacterial plate count of mice were evaluated. In addition, myeloperoxidase (MPO) was detected to assess the infiltration of polymorphonuclear neutrophil (PMN). Cytokine levels were measured by Real Time-PCR and ELISA. Meanwhile, the bacterial burden was also evaluated. The activation of NF-κB signaling was determined by pIκBα/IκBα levels based on Western blot and NF-κB-dependent Luciferase activity on the basis of Luciferase assays using 293T cells. RESULTS: NLRP12 mRNA and protein levels were decreased in B6 corneas and BMDMs after P. aeruginosa infection. The over-expression of NLRP12 in B6 corneas significantly ameliorated the severity of corneal disease, bacterial burden, PMN infiltration and pro-inflammatory cytokine expression. In vitro analysis demonstrated that the up-regulation of NLRP12 suppressed pro-inflammatory cytokine production and enhanced bacterial clearance in RAW264.7 cells. The protein levels of pIκBα and IκBα were significantly decreased after NLRP12-lentivirus treatment compared with that of Ctl-lentivirus. NF-κB-dependent Luciferase activity was potently inhibited by NLRP12 infected with P. aeruginosa or cotransfected with the downstream signaling molecules including IKKα and IKKß in 293T cells. CONCLUSIONS: NLRP12 decreases the severity of P. aeruginosa keratitis, reduces corneal inflammation and bacterial burden through the down-regulation of the NF-κB signaling pathway.

MiRNA regulates OCT4 expression in breast cancer cells.

OBJECTIVE: Breast cancer cell infiltration, migration, and proliferation significantly affect its curative effect. Stemness gene octamer-binding transcription factor 4 (OCT4) upregulated in breast cancer tissue compared with normal control. MiRNA exhibits regulatory role in gene expression. This study adopted bioinformatics to predict the miRNA to regulate OCT4 gene and investigated its impact on breast cancer cell infiltration, migration, and proliferation. MATERIALS AND METHODS: MirBase database was analyzed to explore the potential miRNA in regulating OCT4 based on human OCT4 gene sequence. MiRNA mimics and inhibitor were synthetized and transfected to BS524 cells. qRT-PCR was applied to test miRNA and OCT4 mRNA expressions in cells at 12 h, 24 h, and 48 h after transfection. Western blot was selected to detect OCT4 protein expression. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was selected to determine cell proliferation. Scratch assay was adopted to evaluate cell migration. Transwell assay was used to analyze cell infiltration. RESULTS: MiR-145 may regulate OCT4 gene with score 82. OCT4 mRNA and protein increased at 12 h after transfection (p > 0.05). OCR4 gene significantly upregulated, cell proliferation, migration, and infiltration enhanced by miR-145 transfection compared with control (p < 0.05). OCT4 gene downregulated, while cell proliferation, infiltration, and migration markedly weakened in miR-145 inhibitor group compared with control (p < 0.05). CONCLUSIONS: MiR-145 affects breast cancer BS524 cell proliferation, infiltration, and migration via positively regulating OCT4 gene expression.