ABSTRACT
OBJECTIVE@#To investigate whether circular RNA circRSF1 regulates radiation-induced inflammatory phenotype of hepatic stellate cells (HSCs) by binding to HuR protein and repressing its function.@*METHODS@#Human HSC cell line LX2 with HuR overexpression or knockdown was exposed to 8 Gy X-ray irradiation, and the changes in the expression of inflammatory factors (IL-1β, IL-6 and TNF-α) were detected by qRT-PCR. The expressions of IκBα and phosphorylation of NF-κB were detected with Western blotting. The binding of circRSF1 to HuR was verified by RNA pull-down assay and RNA-binding protein immunoprecipitation (RIP). The expressions of inflammatory factors, IκBα and the phosphorylation of NF-κB were detected after modifying the interaction between circRSF1 and HuR.@*RESULTS@#Knockdown of HuR significantly up- regulated the expressions of IL-1β, IL-6 and TNF-α, decreased IκBα expression and promoted NF-κB phosphorylation in irradiated LX2 cells, whereas overexpression of HuR produced the opposite changes (P < 0.05). Overexpression or knockdown of circRSF1 did not significantly affect the expression of HuR. RNA pull-down and RIP experiments confirmed the binding between circRSF1 and HuR. Overexpression of circRSF1 significantly reduced the binding of HuR to IκBα and down-regulated the expression of IκBα (P < 0.05). Overexpression of circRSF1 combined with HuR overexpression partially reversed the up-regulation of the inflammatory factors, down-regulated IκBα expression and increased phosphorylation of NFκB in LX2 cells, while the opposite effects were observed in cells with knockdown of both circRSF1 and HuR (P < 0.05).@*CONCLUSION@#circRSF1 reduces IκBα expression by binding to HuR to promote the activation of NF-κB pathway, thereby enhancing radiation- induced inflammatory phenotype of HSCs.
Subject(s)
Humans , Hepatic Stellate Cells/radiation effects , Interleukin-6 , NF-kappa B , NF-KappaB Inhibitor alpha , Phenotype , RNA , RNA, Circular/metabolism , Tumor Necrosis Factor-alpha , ELAV-Like Protein 1/metabolismABSTRACT
Objective·To investigate the bacteriostasis effect of Mycobacterium tuberculosis (MTB) polyphosphate kinase 2 (PPK2) aptamer on MTB in vitro.Methods·The bioinformatics method was used to analyze the homology of MTB PPK2 and common pathogens of respiratory tract,and the PPK2 phylogenetic tree was constructed.The binding affinity of the PPK2 aptamer to H37Rv,BCG,Mycobacterium smegmatis,Pseudomonas aeruginosa and Acinetobacter baumannii was analyzed by enzyme-linked oligonucleotide assay (ELONA).The PPK2 aptamer was incubated for 24 h in serum and its biological stability in serum was analyzed by agarose gel electrophoresis.The minimum inhibitory concentration (MIC) of the PPK2 aptamer to H37Rv was determined by micro-azure method.H37Rv was inoculated with 1 μmol/L PPK2 aptamer or random sequence on Roche culture medium for 10 d and colony growth status was observed.H37Rv was co-cultured with different concentrations of PPK2 aptamer for 10 d,absorbance at 600 nm was measured by microplate reader.The effect of PPK2 aptamer on the growth of H37Rv was observed.Results·PPK2 phylogenetic tree constructed by bioinformatics analysis showed that PPK2 protein of H37Rv was not closely related to the common pathogens of respiratory tract,and it was relatively close to Pseudomonas aeruginosa.The ELONA assay results showed that the PPK2 aptamer binded selectively to H37Rv.Agarose gel electrophoresis analysis showed PPK2 aptamer in serum was at least stable for 8 h.The MIC of the PPK2 aptamer to H37Rv was 50 nmol/L.The colony growth of Roche culture showed that PPK2 aptamer had an inhibitory effect on H37Rv growth.Growth inhibition test showed that the absorbance at 600 nm of H37Rv showed a decreasing trend with the increase of PPK2 aptamer concentration,which indicated that PPK2 aptamer had an inhibitory effect on H37Rv growth.Conclusion·PPK2 aptamer has good antibacterial activity against H37Rv in vitro.
ABSTRACT
<p><b>OBJECTIVE</b>To investigate the effect of SAHA on the maturation of human dendritic cells (DC) and to explore its underlying mechanism.</p><p><b>METHODS</b>Peripheral blood mononuclear cells (PBMNC) were isolated from human peripheral blood and cultured in RPMI 1640 medium with 100 ng/ml rhGM-CSF and 500 U/ml rhIL-4. In the LPS induced maturation process, dendritic cells treated with or without SAHA were used as test group, and dendritic cells treated without LPS or SAHA were used as control group. DC was observed under inverted microscope. Flow cytometer was used to detect the surface antigen molecules expressed by DC. The mixed lymphocyte culture (MLC) was used to observe the allogeneic lymphocyte stimulation. The NF-κB signaling pathway was detected by electrophoretic mobility shift assay (EMSA).</p><p><b>RESULTS</b>The SAHA could effectively suppress the maturation of DC induced by LPS, the DC treated with SAHA+LPS had immature morphological characteristics; the expression of CD80, CD83 and HLA-DR in SAHA+LPS group and control group were significantly down-regulated as compared with single LPS group (P<0.01); the ability of DC to stimulate the proliferation of allogeneic T lymphocytes in SAHA+LPS group and control group was significantly weaker than that in single LPS group (P<0.01); EMSA results showed that NF-κB activity decreased after SAHA and LPS treatment and was significantly lower than that of single LPS group.</p><p><b>CONCLUSION</b>SAHA can effectively suppress DC maturation induced by LPS and also weaken the ability to stimulate allogeneic T lymphocyte. NF-κB signaling pathway is involved in regulating DC maturation.</p>