Autophagy Markers Induced by Influenza Virus and MUC1 Expressions in Cancer-Derived Cell Lines

Abstract

Influenza virus-induced autophagy is often accompanied by apoptosis and results in cell death in virus-infected cells. It is well known that autophagy is modulated by the mTOR/PI3K/Akt pathway, which plays an important role in the response to the presence of energy sources and external stimuli. This pathway is modulated by mucin 1 (MUC1), which has extracellular and intracellular components and plays an important role in metastasis and chemotherapeutic resistance. In this study, it was aimed to investigate the expression of MUC1 after the inoculation of influenza viruses into the cancer-derived cell cultures and, accordingly, the changes in autophagy markers such as mTOR and LC3B. In this study, MCF-7, HeLa and A-549 cell lines were used which have adenocarcinoma origin. To control the growth of influenza virus in these cells, the MDCK cell line was also inoculated. Centrifuge-enhanced shell-vial cell culture method was used in all experiments. Influenza A (H1N1) pdm09 strain was inoculated into these cell lines then the expressions of viral nucleic acid and cycle threshold (Ct) of MUC1, mTOR, LC3B associated genes were investigated by quantitative real-time reverse transcriptase polymerase chain reaction (qRTPCR) method in the samples taken from the supernatants of all cells at the end of the 48-hour incubation period. To investigate whether these markers were present in cells, after all cells were permeabilized with paraformaldehyde, cell-coated infected coverslips were stained with fluorescent labeled monoclonal antibodies developed against MUC1, mTOR, LC3B and influenza virus antigens. In the examination of fluorescence microscopy, all of the cell cultures (MCF-7, He-La and A-549) infected with influenza virus yielded positive results in terms of LC3B, mTOR and MUC1 monoclonal antibody staining, whereas all of the non-infected cells were found negative. Cycle threshold values of MUC1, LC3B and mTOR associated genes were found to be lower in A-549 cell line inoculated with influenza virus. Although protein expression was demonstrated in MCF-7 and He-La cell lines, similar changes were not detected in the 1/Ct values of genes in the autophagy pathway. The Ct value of the MUC1 gene was found to be higher only in the MCF-7 cell line after inoculation. In conclusion, it was observed that the specific expression pattern for influenza virus-induced autophagy was formed only in the A-549 cell line among the adenocarcinoma cells. It was thought that this relationship could constitute a dataset in further research on lung adenocarcinoma. However, in future studies, the determination of the expression of these genes at the protein level by using further tests will provide better comparison of the results.