The Influence On Autophagy And Apoptosis In HeLa Caused By PORF5 Plasmid Protein Of Chlamydia Trachomatis

Objective:Autophagy and apoptosis are the key factors for determining cell fate and participate in pathological and physiological processes. With the research deepening, important progress on the regulation mechanisms of autophagy and apoptosis has been made, but the specific regulatory mechanisms of autophagy and apoptosis caused by Chlamydia trachomatis remain unclear. In order to provide further insights into Chlamydia trachomatis pathogenic mechanism, this study attempted to explore the influence on autophagy and apoptosis in HeLa cells triggered by pORF5 plasmid protein of Chlamydia trachomatis.Methods:The pDSRed Cl/pORF5 recombinant bacteria were inoculated into LB solid medium for 12 to 16h, then one bacterial colony was selected to be cultured in LB liquid medium at 37℃ for 4 h, then the pDSRed Cl/pORF5 recombinant plasmid was extracted with OMEGA plasmid mini kit, and the pORF5 gene was amplified with specific primers containing Nhel and EcoRl restriction site from pDSRed Cl/pORF5 plasmid. After restriction enzyme digestion and purification, PCR amplification products and DCE (PCDH-CMV-MCS-EF1-COPGFP) vector were ligased with DNA ligase and transformed into DH5a bacteria. DCE-pORF5 recombinant lentivirus plasmid was identified by colony PCR, restriction enzyme digestion and DNA sequencing. Recombinant lentivirus plasmid and packaging plasmid vector were extracted without endotoxin and co-transfected in 293T cells for virus production. Viruses were collected from supernatant and concentrated, the concentration was determined with the multiple proportion dilution method. The DCE lentivirus and DCE-pORF5 recombinant lentivirus were used to infect HeLa cells for 8h and infection rates were observed by fluorescent microscope. Indirect immunofluorescence and Western blot were used to identify the expression of pORF5 protein in stable cell lines DCE-PORF5-HeLa and DCE-HeLa which separated by flow cytometry. The autophagy-related proteins such as LC3 and Becinl were detected by indirect immunofluorescence and Western blot in DCE-PORF5-HeLa and DCE-HeLa after treated with serum starvation. Flow cytometry was used to detect apoptosis rates of DCE-PORF5-HeLa and DCE-HeLa after treated with TNF-a for 6h. DCE-PORF5-HeLa as well as DCE-HeLa was treated with serum starvation and 3-MA respectively for 24h, and the mRNA levels of Bax and Caspase-3 were analyzed by qRT-PCR.Results:1. DCE-pORF5-HeLa and DCE-HeLa stable cell lines were constructed successfully and the purity reached more than 95%.2. Indirect immunofluorescence assays showed that LC3 red fluorescent spots were appeared in both DCE-HeLa and DCE-pORF5-HeLa cells after treated by serum-free for 24h. Quantification was performed and the results were 34.0+2.6 puncta/cell and 97.6±12.1puncta/cell respectively, the number of LC3 puncta in DCE-pORF5-HeLa was increased significantly when compared with DCE-HeLa (P<0.05)3. Western blot results showed that the expression levels of LC3 and Beclinl were increased by 56% and 57.8% in DCE-HeLa, and increased by 87.2% and 76.6% in DCE-pORF5-HeLa after treated by serum-free for 24 h, when compared with non-treatment groups (P<0.01). The expression of LC3 and Beclinl were increased by 53.3%(P<0.01) and 28.7%(P<0.05) in DCE-PORF5-HeLa, when compared with DCE-HeLa after treated by serum-free for 24 h.4. The mRNA levels of Caspase-3 and Bax in DCE-pORF5-HeLa were reduced by 47.6% and 25.7%(P<0.05) respectively when compared with DCE-HeLa after treated with serum-free, and levels of Caspase-3 and Bax in DCE-pORF5-HeLa were reduced by 73.0% and 43.6%(P<0.05) respectively when compared with DCE-HeLa after treated with 3-MA.5. The apoptosis rates of DCE-HeLa and DCE-PORF5-HeLa were within 5%, but the apoptosis rates were reached (62.9±0.8)% and (44.4±6.03)% respectively in two groups after treated with TNF-a. The apoptosis rate of DCE-pORF5-HeLa was reduced by 29.4% when compared with DCE-HeLa (P<0.05).Conclusion:1. Plasmid protein pORF5 could induce autophagy in HeLa cells.2. Plasmid protein pORF5 could inhibit apoptosis in HeLa cells and the mechanism was associated with autophagy.