Observation of the Effect of Changing Mucin 1 (MUC1) Expressions on Autophagy Pathways in Influenza A Virus-Infected Cell Lines
Project no. 21340 · Status: Kapanmış · 2020
Summary
<p>Aim:In this study,the aim is to observe the changes in MUC1 expressions, which is known to have sialic acid residues,and consequently changes in autophagy markers such as mTOR and LC3B. Methods:After the monolayer culturing of all cell lines (MDCK, RD, He-La, MCF-7, A-549), the cells were inoculated with the influenza virus using the Shell-vial method.Following 48h incubation, all cell lines were immunofluorescent dyed,using specific monoclonal antibodies for mTOR,LC3B and MUC1. Before the initiation of dying procedure, the liquid portion of all cell lines were collected and using specific primary/probe sets targeting mTOR, LC3B and MUC1, gene expressions of the pathways were detected via RT-PCR. For all the procedures, negative cells, which were not inoculated with influenza, were used as controls. MDCK cells were used to control the influenza virus production and one sample of each cell line was dyed with the influenza monoclonal antibody. Results:Inclusion bodys were detected for LC3B, mTOR and MUC-1 in all cell lines inoculated with influenza except for RD.No inclusion body was detected in MCF-7, He-La, RD and A-549 that were not inoculated with influenza by immunofluorescence method.In the supernatant of all influenza-inoculated cell cultures, except for RD cells, positive results were obtained for MUC-1, LC3B and mTOR (Ct 20,01-32,04).In cells without influenza inoculation, only in the A-549 cells the pathways were found to be negative.In other cells,positive results were obtained (Ct 21,98-23,78).In RD cells, which haven’t been inoculated with the virus, all pathways were positive. Conclusion:In cells with adenocarcinoma structure,the presence of autophagy pathways activated via influenza viruses were detected. However, this hasn’t been proved using nucleic acid assays.The reason why might be the detection of only gene expressions and mRNA presence via the tests used in the study.In future studies, expressions of these genes in protein level should be detected using more advanced tests.</p>