ABSTRACT
Objective: To investigate the role of X-box binding protein 1 (XBP1) in the treatment of human osteosarcoma HOS cells by pyropheophorbide-α methyl ester-mediated photodynamic therapy (MPPα-PDT), and to explore the possible mechanism. Methods: HOS cells were treated by MPPα-PDT for 6, 12 and 24 h, and the expression levels of inositol-requiring enzyme 1 alpha (IRE1α)-XBP1 pathway-related protein IRE1α and XBP1 were detected by Western blotting. The specific siRNA targeting XBP1 gene was transfected into HOS cells by lipofection, and treated by MPPα-PDT. The cells were divided into the blank group, siRNA-negative control (NC) group, siRNA-XBP1 group, MPPα-PDT group and MPPα-PDT+siRNA-XBP1 group. The expressions of XBP1 mRNA and protein were detected by real-time fluorescent quantitative PCR and Western blotting, respectively. The proliferation of HOS cells was detected by CCK-8 assay. The apoptotic rate was analyzed by flow cytometry (FCM). The expression levels of cleaved-caspase 3 and cleaved-poly ADP-ribose polymerase (cleaved-PARP) were determined by Western blotting. The intracellular level of reactive oxygen species (ROS) was detected by DCFH-DA probe staining, then the expression levels of Catalase and superoxide dismutase 1 (SOD1) proteins were detected by Western blotting. Results: The expression levels of IRE1α-XBP1 pathway-related proteins IRE1α and XBP1 were increased after the treatment of MPPα-PDT (both P < 0.05 ). siRNA-XBP1 inhibited the expression levels of XBP1 mRNA and protein (both P < 0.01). Silencing siRNA-XBP1 expression inhibited the proliferation activity of HOS cells (P < 0.01), up-regulated the apoptosis rate and the expression level of apoptosis-related protein cleaved-caspase 3 (both P < 0.05), and down-regulated the expression levels of antioxidant enzyme-related proteins Catalase and SOD1 (both P < 0.05). Compared with the MPPα-PDT group, the proliferation activity of HOS cells treated with MPPα-PDT+siRNA-XBP1 was decreased (P < 0.01), the apoptosis rate and the expression levels of cleaved-caspase 3 and cleaved-PARP were increased (all P < 0.05), the intracellular ROS level was up-regulated (P < 0.01), and the expression levels of Catalase and SOD1 were decreased (both P < 0.01). Conclusion: MPPα-PDT can induce the activation of IRE1α-XBP1 pathway in HOS cells. Silencing XBP1 can inhibit the proliferation activity of HOS cells, up-regulate the apoptosis rate, and increase the sensitivity of HOS cells to MPPα-PDT. The mechanism may be related to the up-regulation of intracellular ROS level and the down-regulation of anti-oxidation molecules.
ABSTRACT
Objective To establish a rapid and accurate method of fluorescent quantitative PCR detection for Avian Influenza Virus H7N9 type.Methods A pair of primers and TaqMan fluorescent probes were designed specificly for H7N9 gene ac-cording to the published nucleotide sequence from National Center for Biotechnology Information.Positive recombinant plas-mid was built and the minimum copies of detection,repeatability and specificitv were tested.Results The results showed that this assay obtained positive recombinant plasmid,the linear relation was fine in the range from 102 to 108 copies/μl.The minimum copies was 500 copies/μl with this method,specificitv and repeatability(CV<1%)were fine,no cross reaction with other viruses.The precision was 100% (16/16)with this method for detecting 8 positive samples and 8 negative sam-ples.Conclution The establishment methods of fluorescent quantitative PCR detection could be used for the rapid diagnosis Avian Influenza virus H7N9 types.