Effect Of TGM2 On Tumorigenic Function Of MDCK Cells

Influenza virus is the main cause of human acute respiratory infection,and vaccination is the best way to prevent influenza virus infection.MDCK cells have attracted much attention in the vaccine industry because of their high virus yield and not easy to mutate.Using MDCK cells as virus amplification matrix for influenza vaccine production has many advantages,but its tumorigenicity is still controversial.Tissue transglutaminase 2(TGM2)is a ubiquitous multifunctional protein,which plays an important role in cell adhesion and migration,and is related to the formation of tumor.There is evidence that TGM2 is involved in the interaction between host cells and viruses,but there is no report on whether TGM2 is involved in the replication regulation of influenza viruses.Although the existing production technology of cell matrix inactivated influenza vaccine can ensure the sufficient safety of the vaccine,there is still some controversy about MDCK as the cell matrix for vaccine production because MDCK cells have certain tumorigenicity in nude mice.Experiments show that subcutaneous or intramuscular inoculation of tumor cell lines or their lysates can cause tumors in nude mice.Therefore,whether MDCK cells can be used in human vaccine production still needs to be cautious.At present,the research on the tumorigenicity mechanism of MDCK cells,the use of gene editing to intervene in the formation of cell tumors and ensure that the cell line can be safely used in the production of influenza vaccine is the focus of current research.In previous studies,we found that the expression of TGM2 was significantly up-regulated in many non tumorigenic MDCK cells.Therefore,to explore whether TGM2 affects the tumorigenicity of MDCK cells and whether this gene is suitable as the target gene of MDCK cell tumorigenesis is the main scientific problem to be studied in this paper.In this study,TGM2 overexpression cell lines and TGM2 knockout cell lines were constructed by lentivirus transfection and CRISPR / cas9 technology.The migration and invasion of MDCK cells were studied in vitro and nude mice.At the same time,the proliferation of MDCK cells,the adhesion function between MDCK cells and extracellular matrix and the proliferation level of intracellular influenza virus were concerned.The main results are as follows:(1)The results of scratch and Transwell chamber experiments show that TGM2 overexpression can significantly inhibit the migration and invasion of MDCK cells,and TGM2 knockout can significantly enhance the migration and invasion of MDCK cells.The results of tumorigenesis experiment in nude mice were consistent with those in vitro.TGM2 knockout could significantly enhance the tumorigenesis rate of MDCK cells in nude mice.(2)The migration and invasion ability of rotating cells were verified by scratch test and Transwell chamber.It was found that the invasion ability of tgm2-ko /tgm2-oe cells was significantly lower than that of vector cells.This showed that the TGM2 gene rotation significantly inhibited the migration and invasion of TGM2 knockout cells.These results suggest that TGM2 negatively regulates the tumorigenesis of MDCK cells.(3)The effect of TGM2 on the proliferation of MDCK cells was analyzed by digestion counting method and plate cloning method.It was found that the proliferation level of MDCK cells was significantly up-regulated after TGM2 knockout,and TGM2 overexpression significantly inhibited the proliferation of MDCK cells.It is proved that TGM2 negatively regulates the proliferation of MDCK cells.(4)CCK8 method,fluorescence micrograph counting and crystal violet staining were used to observe the adhesion function of TGM2 overexpression and TGM2 knockout on MDCK cells to extracellular matrix.The experimental results showed that the number of adherent cells of TGM2 overexpression cells was significantly lower than that of vector control cells.The number of adherent cells of TGM2 knockout cells was significantly higher than that of wild-type cells.This indicates that TGM2 can negatively regulate the adhesion between MDCK cells and extracellular matrix.(5)The titer of H1N1 influenza A virus was detected by TCID50 method.The results showed that the titer of H1N1 influenza A virus in MDCK cells increased significantly after TGM2 knockout.TGM2 overexpression significantly reduced the titer of H1N1 influenza A virus,which further proved that TGM2 could inhibit the proliferation of H1N1 influenza A virus in MDCK cells.(6)Through the analysis of gene expression downstream of influenza virus response signal pathway,it was found that the expression levels of Mx1 and ISG15 were significantly up-regulated in TGM2 overexpression cells and down regulated in knockout cells.IFN-α 、 IL-1,IL-8 and IL-6 were significantly down-regulated in overexpressed cells and up-regulated in knockout cells.This suggests that TGM2 may be involved in the regulation of H1N1 influenza A virus proliferation by inhibiting RIG-1 signal pathway and activating JAK-STAT signal pathway.